Methods for treating eye disorders

ABSTRACT

The present invention relates to compositions and methods for inhibiting loss of a retinal ganglion cell in a subject, comprising non-invasively applying to the surface of the eye of the subject an ophthalmic composition comprising a therapeutically effective amount of at least one siRNA which down regulates expression of a target gene associated with loss of the retinal ganglion cell, thereby inhibiting loss of the retinal ganglion cell in the subject. The methods of the invention also relate to the use of chemically modified siRNA compounds possessing structural motifs which down-regulate the expression of human genes expressed in retinal tissue in the mammalian eye.

RELATED APPLICATION

This application is a divisional of application Ser. No. 13/062,161,filed Oct. 22, 2009, which is the National Stage of InternationalApplication No. PCT/US2009/061570, filed Oct. 22, 2009, and which claimsthe benefit of International Application No. PCT/IL2009/000179, filedFeb. 15, 2009 and Provisional Applications No. 61/198,931, filed Nov.11, 2008 and Provisional Application No. 61/196,995, filed Oct. 22,2008, all of which are hereby incorporated by reference herein in theirentirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 17, 2014, isnamed QUARK0008DV.txt and is 5935 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to a non-invasive method of treating aneye disorder in a subject in need thereof comprising the step oftopically administering to the surface of the eye of the subject apharmaceutical composition comprising a therapeutic oligonucleotidedirected to a target gene associated with loss of the retinal ganglioncell in the retina of the subject. Furthermore, the invention relates tonon-invasive method of promoting retinal ganglion cell survival insubject suffering from an ocular disease, disorder or injury.

BACKGROUND OF THE INVENTION

Delivery of nucleic acid compounds to retinal tissue and in particularto retinal ganglion cells presents a big drug delivery challenge.Hitherto, eye drops have been considered to be useful primarily in thetreatment of anterior segment disorders since it is has been shown thatnucleic acids do not pass the cornea and insufficient drugconcentrations reach the posterior ocular tissue (reviewed in del Amoand Urtti, 2008. Drug Discov Today 13(3/4):135-143; Fattal and Bochot,2006. Adv Drug Del Rev 56:1203-1223).

A retinal ganglion cell (RGC) is a type of neuron located near the innersurface of the retina of the eye. Retinal ganglion cells receive visualinformation from photoreceptors and collectively transmit visualinformation from the retina to several regions in the brain.

Further, there remains need for non-invasive method for inhibiting lossof retinal ganglion cells in a subject in need thereof. Various oculardiseases and disorders are characterized by death of retinal ganglioncells (RGCs). Accordingly, there remains a need for a non-invasivemethod for inhibiting loss of retinal ganglion cells (RGCs) in subjectsthat are suffering from an ocular disease, an ocular disorder or anocular injury or are at risk of developing an ocular disease, an oculardisorder, or an ocular injury characterized and/or mediates bydegeneration or death of retinal ganglion cells (RGCs).

SUMMARY OF THE INVENTION

The present invention is directed to non-invasive methods of treatingocular diseases, disorders and injuries that are associated withdegeneration or death of retinal ganglion cells (RGCs) and tocompositions useful in the methods. The methods of the inventioncomprise topically administering to the surface of the eye of a subjecta therapeutic oligonucleotide composition useful in promoting survivalof retinal ganglion cells in a subject. Hitherto, oligonucleotides havebeen delivered to retinal ocular tissue by systemic delivery orintravitreal injection, methods associated with detrimental side effectsand poor patient compliance, respectively. The present inventionprovides topical ophthalmic compositions comprising an oligonucleotide,and non-invasive methods of use thereof for down regulating expressionof a target gene associated with loss of the retinal ganglion cell inthe retina of a subject, for rescuing retinal ganglion cells fromapoptosis and for treating eye disorders.

Accordingly, in one aspect the present invention provides a method ofnon-invasive delivery of an oligonucleotide to a retinal tissue in asubject suffering from an eye disorder, disease or injury comprisingtopically applying an ophthalmic composition comprising theoligonucleotide to the surface of the eye of the subject.

In another aspect the present invention provides a method ofnon-invasive delivery of an oligonucleotide to a retinal ganglion cellin a subject suffering from an eye disorder comprising topicallyapplying an ophthalmic composition comprising the oligonucleotide to thesurface of the eye of the subject.

In yet another aspect, the present invention provides a method ofattenuating expression of a target gene associated with loss of aretinal ganglion cell in the retina in a subject suffering from anocular disease, disorder or injury, which comprises topically(non-invasively) administering to the surface of the eye of the subjecta pharmaceutical composition comprising at least one oligonucleotidedirected to the target mRNA product of the target gene, in an amount andover a period of time effective to attenuate expression of the gene inthe retina of the subject.

In a further aspect, the present invention provides a method of treatinga subject suffering from retinal ganglion cell loss or retinal ganglioncell damage and providing ocular neuroprotection to a subject sufferingfrom or at risk of developing an eye disease, disorder or injury. Themethod comprises topically administering to the surface of the eye ofthe subject an ophthalmic pharmaceutical composition comprising at leastone oligonucleotide directed to a target gene in the retina of thesubject, in an amount and over a period of time effective to inhibitretinal ganglion cell loss or retinal ganglion cell damage in thesubject.

In various embodiments the ophthalmic pharmaceutical composition isformulated as a cream, a foam, a paste, an ointment, an emulsion, aliquid solution including an eye drop, a gel, spray, a suspension, amicroemulsion, microspheres, microcapsules, nanospheres, nanoparticles,lipid vesicles, liposomes, polymeric vesicles, a patch, or a contactlens. In some embodiments the pharmaceutical composition is formulatedas an eye drop. In preferred embodiments the ophthalmic composition isadministered to the eye, for example, by instillation of an eye drop orby administration of a mist.

In certain embodiments the at least one target ocular mRNA is selectedfrom an mRNA polynucleotide set forth in any one of SEQ ID NOS:1-58. Incertain embodiments the at least one target gene is selected from a genetranscribed into an mRNA polynucleotide set forth in any one of SEQ IDNOS:1-58.

In some embodiments the at least one oligonucleotide is selected fromchemically modified siRNA, unmodified siRNA, antisense, ribozyme, miRNAand shRNA. In preferred embodiments the at least one oligonucleotide isa chemically modified siRNA. In some embodiments the siRNA sense andantisense oligonucleotides are selected from sense and correspondingantisense oligonucleotide pairs shown in Tables B1-B36, set forth in SEQID NOS:59-33,596.

In some embodiments the eye disorder, disease or injury is selected fromglaucoma, dry eye, diabetic retinopathy (DR), diabetic macular edema(DME) or age related macular degeneration (AMD). In other embodimentsthe ocular disorder, disease or injury is optic neuritis, centralretinal vein occlusion, brunch retinal vein occlusion (BRVO). In furtherembodiments the eye disorder, disease or injury is retinitis pigmentosa(RP), ischemic optic neuropathy or optic nerve injury. In furtherembodiments ocular disorder, disease or injury is retinopathy ofprematurity (ROP) retinal ganglion degeneration, macular degeneration,hereditary optic neuropathy, metabolic optic neuropathy, opticneuropathy due to a toxic agent or neuropathy caused by adverse drugreactions or vitamin deficiency. In yet another embodiment the disorderis vision loss associated with a tumor.

In various embodiments the at least one siRNA compound is delivered tothe eye of a subject as a liquid solution, including an eye drop. Invarious embodiments the present invention provides a method forattenuating expression of a target ocular gene associated with loss of aretinal ganglion cell in a subject suffering from an ocular disease,disorder or injury, comprising topically applying to the surface of theeye of the subject an ophthalmic pharmaceutical composition formulatedas an eye drop.

In various embodiments expression of a target ocular mRNA is attenuatedin an ocular cells in the retina of a subject suffering from an eyedisease, disorder, or injury. In various embodiments the ocular cellincludes but is not limited to an acinar cell of the lacrimal gland, aductal cell of the lacrimal gland, a retinal ganglion cell (RGC), aretinal pigment epithelial (RPE) cell, a choroidal cell, a corneal cell,a cell of the ciliary process or a cell of the trabecular meshwork or acombination thereof.

The present invention provides a method for inhibiting loss of a retinalganglion cell in a subject, comprising non-invasively administering tothe surface of the eye of the subject an ophthalmic compositioncomprising a therapeutically effective amount of at least one siRNAwhich down regulates expression of a target gene associated with loss ofthe retinal ganglion cell in the retina of the subject, therebyinhibiting loss of a retinal ganglion cell. In certain embodiments twoor more target genes are down regulated by the method according to theinvention. In certain embodiments attenuating expression of at least onetarget gene (ocular mRNA) associated with loss of the retinal ganglioncell in the retina of the subject confers upon the eye neuroprotectiveproperties. In various embodiments the at least one ocular target geneis selected from the list in Tables A1 to A4, set forth in SEQ IDNO:1-58.

In one embodiment the eye disorder is glaucoma. Thus the presentinvention provides a method of inhibiting loss of a retinal ganglioncell in a subject suffering from glaucoma comprising topically(non-invasively) applying to the surface of the eye of the subject anophthalmic composition comprising a therapeutically effective amount ofat least one siRNA which down regulates a target gene (target ocularmRNA) associated with loss of the retinal ganglion cell in the retina ofthe subject, thereby inhibiting loss of a retinal ganglion cell in thesubject's eye. In preferred embodiments the siRNA is chemicallymodified. In some embodiments the chemically modified siRNA attenuatesexpression of gene (target ocular mRNA) in the retina of the subject'seye. In other embodiments the chemically modified siRNA attenuatesexpression of target ocular mRNA in the optic nerve of the subject'seye. In certain embodiments attenuating expression of at least onetarget gene (ocular mRNA) associated with loss of the retinal ganglioncell in the retina of the subject is effective to treat glaucoma. Incertain embodiments the at least one target gene is selected from thelist in Table A1 set forth in SEQ ID NO:1-35. In certain preferredembodiments the target gene is selected from CASP2 (SEQ ID NO:1-2),ASPP1 (SEQ ID NO:4), TP53BP2 (SEQ ID NO:6-7), BNIP3 (SEQ ID NO:12),RTP801L (SEQ ID NO:14), ACHE (SEQ ID NO:19-20), ADRB1 (SEQ ID NO:21) andCAPNS1 (SEQ ID NO:28-29). In various embodiments the gene is set forthin SEQ ID NOS:1-2. In some embodiments the siRNA sense and antisensestrands are selected from pairs of oligonucleotide sequences set forthin SEQ ID NOS:8515-9516.

In another embodiment the eye disorder is dry-eye. Thus the presentinvention provides a method of inhibiting loss of a retinal ganglioncell in a subject suffering from dry-eye. The method comprises topically(non-invasively) administering to the surface of the eye of the subjectan ophthalmic composition comprising a therapeutically effective amountof at least one siRNA which down regulates a target gene (target ocularmRNA) associated with loss of the retinal ganglion cell in the retina ofthe subject, thereby inhibiting loss of a retinal ganglion cell in thesubject's eye. In preferred embodiments the siRNA is chemicallymodified. In certain embodiments attenuating expression of at least onetarget ocular mRNA is effective to treat dry-eye disorder. In certainembodiments attenuating expression of at least one target geneassociated with loss of retinal ganglion cells is effective to reducethe symptoms of dry-eye. In certain embodiments the at least one targetgene (ocular target mRNA) is expressed in retinal ganglion cell in theeye of the subject. In certain embodiments the at least one target gene(ocular target mRNA) is expressed in the lacrimal gland in the eye ofthe subject. In certain embodiments the at least one target gene isselected from Table A2 set forth in any one of SEQ ID NO: 5 (p53), SEQID NO:8-10 (LRDD), SEQ ID NO:26-27 (SHC1) and SEQ ID NO:30-44 (FAS andFAS ligand). In some embodiments the target gene is selected from FAS,FAS ligand (FASL), p53, LRDD, PARP1, AIF (apoptosis inducing factor),NOS1, NOS2A, XIAP and SHC1-SHC. In certain preferred embodiments thetarget gene is set forth in any one of SEQ ID NO:36-SEQ ID NO: 43 or SEQID NO:44. In some embodiments the siRNA sense and antisense strands areselected from pairs of oligonucleotide sequences set forth in SEQ IDNOS: 13,225-15,224.

In another embodiment the eye disorder is AMD, DR or DME. Thus, thepresent invention provides a method of inhibiting loss of a retinalganglion cell in a subject suffering from AMD, DR or DME. The methodcomprises topically (non-invasively) administering to the surface of theeye of the subject an ophthalmic composition comprising atherapeutically effective amount of at least one siRNA which downregulates a target gene (target ocular mRNA) associated with loss of theretinal ganglion cell in the retina of the subject, thereby inhibitingloss of a retinal ganglion cell in the subject's eye. In preferredembodiments the siRNA is chemically modified. In certain embodimentsattenuating expression of at least one target gene associated with lossof retinal ganglion cells is effective to treat AMD, DR or DME. Incertain embodiments attenuating expression of at least one target geneassociated with loss of retinal ganglion cells is effective to reducethe symptoms of AMD, DR or DME. In certain embodiments theadministration of at least one siRNA attenuates expression of at leastone target gene (ocular mRNA) in a retinal ganglion cell in thesubject's eye. In certain embodiments the administration of at least onesiRNA attenuates expression of at least one target gene (target ocularmRNA) in the choroid in the subject's eye. In certain embodiments the atleast one target gene is listed in Table A3 set forth in any one of SEQID NOS:1-2, 3, 5, 6-7, 8-10, 12, 13, 24-25, 26-27, 30-35, 45-53. Incertain preferred embodiments the siRNA targets CTSD, RTP801 and BNIP3.In certain preferred embodiments the disorder is DR and the siRNAtargets mRNA set forth in SEQ ID NOS:48-53. In some embodiments thesiRNA sense and antisense strands are selected from any one of sequencesset forth in SEQ ID NOS:24575-29594. In certain preferred embodimentsthe disorder is AMD and the siRNA targets gene set forth in SEQ ID NO:3and the siRNA sense and antisense strands are selected from any one ofsequences set forth in SEQ ID NOS:11285-12224. In some embodiments thesiRNA sense and antisense strands are selected from pairs ofoligonucleotide sequences set forth in SEQ ID NOS:24575-29594.

In a further embodiment the eye disorder is Retinitis pigmentosa (RP).Thus, the present invention provides a method of inhibiting loss of aretinal ganglion cell in a subject suffering from RP. The methodcomprises topically (non-invasively) administering to the surface of theeye of the subject an ophthalmic composition comprising atherapeutically effective amount of at least one siRNA which downregulates a target gene (target ocular mRNA) associated with loss of theretinal ganglion cell in the retina of the subject, thereby inhibitingloss of a retinal ganglion cell in the subject's eye. In preferredembodiments the siRNA is chemically modified. In certain embodimentsattenuating expression of at least one target gene associated with lossof retinal ganglion cells is effective to RP. In certain embodimentsattenuating expression of at least one target gene associated with lossof retinal ganglion cells is effective to reduce the symptoms of RP. Incertain embodiments the at least one target ocular mRNA is product of agene selected from a gene listed in Table A4 which is transcribed intomRNA set forth in any one of SEQ ID NOS: 3, 14, 26-35, 54-57. In someembodiments the target ocular mRNA is product of a gene selected fromthe group consisting of CASP1, CASP3, CASP12, RTP801, RTP801L, CAPNS1,PARP1, AIF, NOS1, NOS2, XIAP and SHC1-SHC. In certain preferredembodiments the siRNA targets mRNA set forth in SEQ ID NOS:56-57.

In another aspect the invention features a method of rescuing a retinalganglion cell from apoptosis in a subject, comprising non-invasivelyapplying to the surface of the eye of the subject an ophthalmiccomposition comprising a therapeutically effective amount of at leastone siRNA to a target gene in the retina of the subject, therebyrescuing retinal ganglion cell from apoptosis in the subject. Inpreferred embodiments the at least one siRNA is a chemically modified.In some embodiments the target gene is set forth in any one of SEQ IDNO:1-58. In various embodiments the siRNA sense and antisenseoligonucleotides are selected from sense and corresponding antisenseoligonucleotides shown in tables B1-B36 and set forth in SEQ IDNOS:59-33,596.

In another aspect, the invention provides method for promoting survivalof a retinal ganglion cell in a subject displaying signs or symptoms ofan ocular neuropathy, comprising non-invasively administering to thesurface of the eye of the subject an ophthalmic composition comprising atherapeutically effective amount of at least one siRNA to a target genein the retina of the subject, thereby promoting survival of a retinalganglion cell in the subject. In preferred embodiments the at least onesiRNA is a chemically modified. In certain embodiments the signs orsymptoms of an ocular neuropathy are mediated by apoptosis. In someembodiments the target gene is set forth in any one of SEQ ID NO:1-58.In various embodiments the siRNA sense and antisense oligonucleotidesare selected from sense and corresponding antisense oligonucleotides setforth in SEQ ID NOS:59-33,596.

In yet another aspect, the invention is directed to a method forpreventing, treating or alleviating the effects of an ocular diseaseassociated with death of a retinal ganglion cell in a subject,comprising non-invasively applying to the surface of the eye of thesubject an ophthalmic composition comprising a therapeutically effectiveamount of at least one siRNA to a target gene in the retina of thesubject, thereby preventing, treating or alleviating the effects of anocular disease associated with death of a retinal ganglion cell in thesubject. In preferred embodiments the at least one siRNA is a chemicallymodified. In some embodiments the target gene is set forth in any one ofSEQ ID NO:1-58. In various embodiments the siRNA sense and antisenseoligonucleotides are selected from sense and corresponding antisenseoligonucleotide pairs shown in Tales B1-B36, set forth in SEQ IDNOS:59-33,596.

Another aspect of the invention provides for a method for treating orpreventing retinal ganglion cell death in a subject, comprisingnon-invasively applying to the surface of the eye of the subject anophthalmic pharmaceutical composition which comprises: (a) atherapeutically effective amount of at least one siRNA to a target genein the retina of the subject, and (b) a pharmaceutically acceptableexcipient or carrier or mixture thereof, and thereby treating orpreventing retinal ganglion cell death in the subject. In preferredembodiments the at least one siRNA is a chemically modified. In someembodiments the target gene is set forth in any one of SEQ ID NO:1-58.In various embodiments the siRNA sense and antisense oligonucleotidesare selected from sense and corresponding antisense oligonucleotides setforth in SEQ ID NOS:59-33,596.

In another aspect, the present invention is directed to a methodpreventing retinal ganglion cell death mediated by elevated intraocularpressure (IOP) in the eye of a subject, comprising non-invasivelyadministering to the surface of the eye of the subject an ophthalmiccomposition comprising a therapeutically effective amount of at leastone siRNA to a target gene in the retina of the subject, therebypreventing retinal ganglion cell death in the subject. In preferredembodiments the at least one siRNA is a chemically modified. In aparticular embodiment according to this method the subject is afflictedwith glaucoma. In some embodiments the target gene is set forth in anyone of SEQ ID NO:1-58. In various embodiments the siRNA sense andantisense oligonucleotides are selected from sense and correspondingantisense oligonucleotides set forth in SEQ ID NOS:59-33,596.

The present invention further provides a method of delaying, preventingor rescuing a retinal cell from death in a subject suffering fromelevated IOP comprising non-invasively applying to the surface of theeye of the subject an ophthalmic composition comprising atherapeutically effective amount of at least one siRNA to a target geneassociated with death of the RGC in the retina of the subject, therebydelaying, preventing or rescuing the retinal cell from injury or deathand wherein intraocular pressure (IOP) remains substantially elevated.In preferred embodiments the at least one siRNA is a chemicallymodified. In a particular embodiment according to this method thesubject is afflicted with glaucoma. In some embodiments the target geneis set forth in any one of SEQ ID NO:1-58. In various embodiments thesiRNA sense and antisense oligonucleotides are selected from sense andcorresponding antisense oligonucleotides set forth in SEQ IDNOS:59-33,596.

The present invention further provides a method of treating a subjectsuffering from retinal ganglion cell loss or retinal ganglion celldamage, comprising non-invasively administering to the surface of theeye of the subject an ophthalmic composition comprising atherapeutically effective amount of at least one siRNA to a target genein the retina of the subject, thereby treating the subject or reducingretinal ganglion cell death in the subject. In preferred embodiments theat least one siRNA is a chemically modified. In some embodiments thetarget gene is set forth in any one of SEQ ID NO:1-58. In variousembodiments the siRNA sense and antisense oligonucleotides are selectedfrom sense and corresponding antisense oligonucleotides set forth in SEQID NOS:59-33,596.

The present invention further provides a method for lowering retinalganglion cell loss and providing ocular neuroprotection to a subject inneed thereof, comprising non-invasively applying to the surface of theeye of the subject an ophthalmic composition comprising atherapeutically effective amount of at least one siRNA to a target genein the retina of the subject, thereby lowering retinal ganglion cellloss and providing ocular neuroprotection to the subject. In preferredembodiments the at least one siRNA is a chemically modified. In someembodiments the target gene is set forth in any one of SEQ ID NO:1-58.In various embodiments the siRNA sense and antisense oligonucleotidesare selected from sense and corresponding antisense oligonucleotidepairs shown in Tables B1-B36, set forth in SEQ ID NOS:59-33,596.

The present invention further provides a method for preventing visualfield loss associated with loss of retinal ganglion cells in a subject,comprising non-invasively administering to the surface of the eye of thesubject an ophthalmic composition comprising a therapeutically effectiveamount of at least one siRNA to a target gene in the retina of thesubject, thereby preventing visual field loss in the subject. Inpreferred embodiments the at least one siRNA is a chemically modified.In some embodiments the target gene is set forth in any one of SEQ IDNO:1-58. In various embodiments the siRNA sense and antisenseoligonucleotides are selected from sense and corresponding antisenseoligonucleotide pairs shown in Tables B1-B36, set forth in SEQ IDNOS:59-33,596.

In another aspect, the present invention provides an ophthalmiccomposition for non-invasive treatment of an ocular disease associatedwith loss of a retinal ganglion cell in a subject, comprising: (a) atherapeutically effective amount of at least one siRNA to a target genein the retina of the subject, and (b) a pharmaceutically acceptableexcipient or carrier or mixture thereof. In preferred embodiments the atleast one siRNA is a chemically modified. In some embodiments the targetgene is set forth in any one of SEQ ID NO:1-58. In various embodimentsthe siRNA sense and antisense oligonucleotides are selected from senseand corresponding antisense oligonucleotide pairs shown in TablesB1-B36, set forth in SEQ ID NOS:59-33,596.

In yet another aspect, the present invention provides a topicalophthalmic pharmaceutical composition for non-invasive treatment of anocular disease associated with pathological abnormalities/changes in thetissues of the visual system, comprising: (a) a therapeuticallyeffective amount of at least one siRNA to a target gene in the retina ofthe subject, wherein the target gene is set forth in any one of SEQ IDNO:1-58, and (b) a pharmaceutically acceptable excipient or carrier ormixture thereof. In preferred embodiments the at least one siRNA is achemically modified. In various embodiments the siRNA sense andantisense oligonucleotides are selected from sense and correspondingantisense oligonucleotide pairs shown in Tables B1-B36, set forth in SEQID NOS:59-33,596.

According to another aspect, the present invention is directed to atopical ophthalmic pharmaceutical composition for use in treating asubject afflicted with ocular disease associated with death of retinalganglion cells, which comprises: (a) a therapeutically effective amountof at least one siRNA to a target gene in the retina of the subject,wherein the target gene is set forth in any one of SEQ ID NO:1-58, and(b) a pharmaceutically acceptable excipient or carrier or mixturethereof. In preferred embodiments the at least one siRNA is a chemicallymodified. In various embodiments the siRNA sense and antisenseoligonucleotides are selected from sense and corresponding antisenseoligonucleotide pairs shown in Tables B1-B36, set forth in SEQ IDNOS:59-33,596.

In various embodiments the ocular disease is selected from a groupcomprising glaucoma, dry eye, diabetic retinopathy (DR), diabeticmacular edema (DME), age related macular degeneration (AMD), opticneuritis, central retinal vein occlusion, brunch retinal vein occlusion,ischemic optic neuropathy, optic nerve injury, retinopathy ofprematurity (ROP) or retinitis pigmentosa (RP), retinal gangliondegeneration, macular degeneration, hereditary optic neuropathy,metabolic optic neuropathy, neuropathy due to a toxic agent or thatcaused by adverse drug reactions or vitamin deficiency; and thecomposition is formulated as a cream, a foam, a paste, an ointment, anemulsion, a liquid solution, an eye drop, a gel, spray, a suspension, amicroemulsion, microspheres, microcapsules, nanospheres, nanoparticles,lipid vesicles, liposomes, polymeric vesicles, a patch, a biologicalinsert. In a preferred embodiment the composition is formulated as aneye drop.

In another aspect the invention is directed to a packaged pharmaceuticalpreparation, comprising: (a) a pharmaceutical composition according tothe invention in a container; and (b) instructions for using thecomposition to treat an ocular disease. In various embodiments thepharmaceutical composition according to present invention comprises atherapeutically effective amount of at least one siRNA to a target genein the retina of the subject suffering from an ocular disease. Inpreferred embodiments the at least one siRNA is a chemically modified.In some embodiments the target gene is set forth in any one of SEQ IDNO:1-58. In various embodiments the siRNA sense and antisenseoligonucleotides are selected from sense and corresponding antisenseoligonucleotides set forth in SEQ ID NOS:59-33,596. In one particularembodiment according to this aspect of the invention, the pharmaceuticalcomposition is for non-invasive treatment of an ocular diseaseassociated with loss of a retinal ganglion cell in a subject. In anotherparticular embodiment according to this aspect the invention, thepharmaceutical composition is for non-invasive treatment of an oculardisease associated with pathological abnormalities/changes in thetissues of the visual system. In yet another particular embodimentaccording to this aspect the invention, the pharmaceutical compositionis for use in treating a subject afflicted with ocular diseaseassociated with death of retinal ganglion cells.

According to another aspect, the invention is directed to use of apharmaceutical composition according to the invention in the manufactureof a medicament for promoting retinal ganglion cell survival in asubject. In various embodiments the pharmaceutical composition accordingto present invention comprises a therapeutically effective amount of atleast one siRNA to a target gene in the retina of the subject sufferingfrom an ocular disease. In preferred embodiments the at least one siRNAis a chemically modified. In some embodiments the target gene is setforth in any one of SEQ ID NO:1-58. In various embodiments the siRNAsense and antisense oligonucleotides are selected from sense andcorresponding antisense oligonucleotide pairs shown in Tables B1-B36,set forth in SEQ ID NOS:59-33,596. In one particular embodimentaccording to this aspect the invention the pharmaceutical composition isfor non-invasive treatment of an ocular disease associated with loss ofa retinal ganglion cell in a subject. In another particular embodimentaccording to this aspect the invention the pharmaceutical composition isfor non-invasive treatment of an ocular disease associated withpathological abnormalities/changes in the tissues of the visual system.In yet another particular embodiment according to this aspect theinvention the pharmaceutical composition is for use in treating asubject afflicted with ocular disease associated with death of retinalganglion cells.

In some embodiments the composition includes a viscosity enhancingagent. A viscosity enhancing agent is selected from for example ahydrophilic polymer including cellulose and cellulose derivativesmethylcellulose and methylcellulose derivatives. Such agents includemethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, carboxymethyl cellulose, derivativesthereof, combinations thereof and their salts. In some embodiments theviscosity enhancing agent is methylcellulose. In certain embodiments theviscosity enhancing agent is provided at a concentration of about 0.01%to about 4%, in other embodiments the viscosity enhancing agent isprovided at a concentration of about 0.1% to about 3%, or at aconcentration of about 0.5% to about 2%.

In some embodiments the composition includes an agent providing osmoticbalance, or a surfactant. Such agents include glycerol, ethylene glycol,poly(ethylene glycol), propylene glycol, sorbitol, mannitol,monosaccharide, disaccharides and oligosaccharides.

The present invention further provides a method of treating glaucoma ina subject in need of treatment that comprises topically (non-invasively)administering to the surface of the eye of the subject a therapeuticallyeffective amount of at least one siRNA to a target gene in the eye ofthe subject, in an amount effective to treat glaucoma. In certainpreferred embodiments the siRNA chemically modified siRNA. In certainembodiments the at least one siRNA inhibits expression of at least onegene expressed in the retina of the subject's eye. In certainembodiments inhibition of at least one gene confers upon the eyeneuroprotective properties. In certain embodiments the at least one geneis selected from a list in Table A1 transcribed into mRNA set forth inSEQ ID NO:1-35. In certain preferred embodiments the gene is selectedfrom CASP2 (SEQ ID NO:1-2), ASPP1 (SEQ ID NO:4), TP53BP2 (SEQ IDNO:6-7), BNIP3 (SEQ ID NO:12), RTP801L (SEQ ID NO:14), ACHE (SEQ IDNO:19-20), ADRB1 (SEQ ID NO:21) and CAPNS1 (SEQ ID NO:28-29). In variousembodiments the gene is set forth in SEQ ID NOS:1-2. In some embodimentsthe siRNA sense and antisense strands are selected from any one ofsequences set forth in SEQ ID NOS:8515-9516.

The present invention further provides a method of treating dry-eye in asubject in need thereof, which comprises topically (non-invasively)applying to the surface of the eye of the subject a therapeuticallyeffective amount of at least one siRNA which inhibits expression of atleast one gene expressed in the eye of the subject in an amounteffective to reduce the symptoms of dry eye. In certain preferredembodiments the siRNA is chemically modified siRNA. In certainembodiments the at least one gene is expressed in the lacrimal gland inthe subject. In certain embodiments the at least gene is selected fromTable A2 set forth in any one of SEQ ID NO: 5, SEQ ID NO:8-10, SEQ IDNO:26-27 and SEQ ID NO:30-44. In some embodiments the gene is selectedfrom FAS, FAS ligand (FASL), p53, LRDD, PARP1, AIF (apoptosis inducingfactor), NOS1, NOS2A, XIAP and SHC1-SHC. In certain preferredembodiments the gene is transcribed into mRNA set forth in any one ofSEQ ID NO:36-SEQ ID NO:44. In some embodiments the siRNA sense andantisense strands are selected from any one of sequences in Tables 16-17set forth in SEQ ID NOS: 13,225-15,224.

The present invention further provides a method of treating AMD, DR orDME in a subject in need thereof, which comprises topically(non-invasively) administering to the surface of the eye of the subjecta therapeutically effective amount of at least one siRNA which inhibitsexpression of at least one gene expressed in the eye in the subject inan amount effective to reduce the symptoms of AMD, DR or DME. In certainpreferred embodiments the siRNA is chemically modified siRNA. In certainembodiments the at least one siRNA inhibits expression of at least onegene expressed in the choroid in the subject's eye. In certainembodiments the at least one target ocular mRNA listed in Table A3, setforth in any one of SEQ ID NOS:1-2, 3, 5, 6-7, 8-10, 12, 13, 24-25,26-27, 30-35, 45-53. In certain preferred embodiments the siRNA targetsCTSD, RTP801 and BNIP3. In certain preferred embodiments the disorder isDR and the siRNA targets mRNA set forth in SEQ ID NOS:48-53.

In a further embodiment the eye disorder is Retinitis pigmentosa (RP).Thus, the present invention provides a method of attenuating expressionof a target ocular mRNA in the eye of a subject suffering from RP. Themethod comprises topically (non-invasively) applying to the surface ofthe eye of the subject a therapeutically effective amount of at leastone siRNA to a target gene in the eye of the subject. In certainpreferred embodiments the siRNA is chemically modified siRNA. In certainembodiments attenuating expression of at least one target gene (targetocular mRNA) is effective to treat RP. In certain embodimentsattenuation of at least one target ocular mRNA is effective to reducethe symptoms of RP. In certain embodiments the at least one targetocular mRNA is product of a gene selected from a gene listed in Table A4which is transcribed into mRNA set forth in any one of SEQ ID NOS: 3,14, 26-35, 54-57. In some embodiments the target ocular mRNA is productof a gene selected from the group consisting of CASP1, CASP3, CASP12,RTP801, RTP801L, CAPNS1, PARP1, AIF, NOS1, NOS2, XIAP and SHC1-SHC. Incertain preferred embodiments the siRNA targets mRNA set forth in SEQ IDNOS:56-57.

In various embodiments the at least one siRNA is chemically modified. Invarious embodiments the at least one siRNA comprises a sufficient numberof consecutive nucleotides having a sequence of sufficient homology to anucleic acid sequence present within the target mRNA to hybridize to themRNA and attenuate expression of the mRNA in the eye of the subject.

In various embodiments the at least one siRNA comprises a sufficientnumber of consecutive nucleotides having a sequence of sufficienthomology to a nucleic acid sequence present within the gene to hybridizeto the gene and reduce or inhibit expression of the gene in the eye ofthe subject.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C: Representative images of Cy3 labeled DDIT4 siRNAincorporated into murine retina following eye drop administration.

FIGS. 2A-2D: Representative images of Cy3 labeled DDIT4 siRNAincorporated into ductal and acinar cells of the murine lacrimal glandfollowing eye drop administration.

FIG. 3A shows accumulation of Cy3-siRNA in rat choroid afteradministration by eye drops 10 minutes post administration.

FIG. 3B shows accumulation of Cy3-siRNA in rat choroid afteradministration by eye drops 1 hour post administration.

FIG. 3C shows accumulation of Cy3-siRNA in rat choroid afteradministration by eye drops 4 hours post administration.

FIG. 4: Cy3-siRNA delivery to the trabecular meshwork and ciliary bodyone hour after eye drop administration to the eye.

FIGS. 5A-5B: Confocal microscopy (magnification ×60) of the retina 1hour after eye drop administration of QM5—siRNA targeted at the p53gene. SiRNA in retina (retinal pigment epithelial cells, retinalganglion cells) is shown by Cy3 fluorescence.

FIGS. 6A-6B are representative images of Cy3 labeled DDIT4 siRNAincorporated into mice retina. FIG. 6B shows accumulation of DDIT4_(—)1Cy3-siRNA 1 hour post administration by eye drops. Choroid, outernuclear layer, RPE and outer segment layer of photoreceptor cells showCy3 staining.

FIG. 7 shows accumulation of DDIT4_(—)1 Dy-649/C6-siRNA 1 hour postadministration by eye drops in RGC cells by use of Dy-649/C6 staining.

FIG. 8A-8C represent control siRNA FITC-CNL_(—)1RD/CNL_(—)1FD andscarmbled3′ cy3-CNL_(—)1 delivery to retinal tissues by differentstaining methods.

FIGS. 9A-9B show delivery of different structures of Casp2 to the mouseretinal tissues.

FIGS. 10A-10B show TGASEII-FAM and HNOEL-FAM delivery.

FIG. 11 shows retinal delivery of siRNA against p53 in PBS as positivecontrol group. FIGS. 12A-12B show that in intact animals or whenadministering ED without siRNA no fluorescent signal is obtained in theretina.

FIG. 13A shows different dilutions of kidney protein extract andmeasurement of p53 protein levels by specific ELISA assay creating astandard curve.

FIG. 13B shows ELISA standard curve based on purified fused GST-hp53protein.

FIG. 13C is a graphical representation of the results provided in TableC14 and depicting the effect of treatments with an siRNA compoundtargeting the p53 gene (QM5), administered intravitreally or byapplication of eye drops, on the expression of p53 as measured inretinal protein extracts using an ELISA test. p53 protein levels in theretina were calculated according to standard curve shown in FIG. 13A.

FIG. 13D is a graphical representation of the results provided in TableC16 and depicting the effect of treatments with an siRNA compoundtargeting the p53 gene (QM5), administered intravitreally or byapplication of eye drops, on expression of p53 as measured in retinalprotein extracts using an ELISA standard curve based on purified fusedGST-hp53 protein. p53 protein levels in the retina were calculatedaccording to standard curve shown in FIG. 13B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides topical oligonucleotide compositions andnon-invasive methods of use thereof for treating various eye diseasesand disorders. In particular, the invention provides methods fortreatment of various eye diseases and disorders associated with geneexpression in the eye of a subject suffering from the disease ordisorder.

The present invention is based in part on the unexpected discovery thattopical non-invasive administration of siRNA compositions targetscertain ocular tissues and cell types and is active in those tissues andcells when delivered topically to the surface of the eye. The discoveryis surprising in view of the known obstacles to siRNA delivery andprovides non-invasive methods as a realistic alternative to intravitrealor systemic delivery.

For siRNA molecules to be effective in silencing mRNA of a target gene,the siRNA requires three levels of targeting: to the target tissue, tothe target cell type and to the target subcellular compartment. Thepresent invention now discloses non-invasive methods of treating eyediseases and disorders.

The present invention relates in general to compounds whichdown-regulate expression of genes expressed in ocular cells,particularly to novel small interfering RNAs (siRNAs), and to the use ofthese novel siRNAs in the treatment of a subject suffering from medicalconditions associated with expression of those genes in eye tissues andcells.

Methods for attenuating expression of an ocular target mRNA and methodsof treating disorders in the eye are discussed herein at length, and anyof said molecules and/or compositions may be beneficially employed inthe treatment of a subject suffering from any of said conditions.

The siRNAs of the present invention possess structures and modificationswhich may increase activity, increase stability, and or minimizetoxicity; the novel modifications of the siRNAs of the present inventioncan be beneficially applied to double stranded RNA sequences useful inpreventing or attenuating target gene expression, in particular thetarget genes discussed herein.

Details of a non-limited example of target genes per indication arepresented in Tables A1-A4, hereinbelow.

TABLE A1 Target genes for treatment of glaucoma Gene Full name and HumanGene ID/SEQ ID NO. for mRNA polynucleotide CASP2 caspase 2,apoptosis-related cysteine peptidase gi|39995058|ref|NM_032982.2 (SEQ IDNO: 1) gi|39995060|ref|NM_032983.2 (SEQ ID NO: 2) RTP801 Homo sapiensDNA-damage-inducible transcript 4 (DDIT4), mRNAgi|56676369|ref|NM_019058.2 (SEQ ID NO: 3) ASPP1 protein phosphatase 1,regulatory (inhibitor) subunit 13B (PPP1R13B)gi|121114286|ref|NM_015316.2 (SEQ ID NO: 4) p53 tumor protein p53gi:120407067, NM_000546.2 (SEQ ID NO: 5) TP53BP2 tumor protein p53binding protein, 2 gi|112799848|ref|NM_001031685.2 (SEQ ID NO: 6)gi|112799845|ref|NM_005426.2 (SEQ ID NO: 7) LRDD leucine-rich repeatsand death domain containing variant 2 gi|61742781|ref|NM_018494.3 (SEQID NO: 8) variant 1 gi|61742783|ref|NM_145886.2 (SEQ ID NO: 9) variant 3gi|61742785|ref|NM_145887.2 (SEQ ID NO: 10) CYBA cytochrome b-245, alphapolypeptide gi|68509913|ref|NM_000101.2 (SEQ ID NO: 11) BNIP3BCL2/adenovirus E1B 19 kDa interacting protein 3gi|7669480|ref|NM_004052.2 (SEQ ID NO: 12) RAC1 ras-related C3 botulinumtoxin substrate 1 (rho family, small GTP binding protein)(gi|38505164|ref|NM_198829.1) (SEQ ID NO: 13) RTP801L Homo sapiensDNA-damage-inducible transcript 4-like (DDIT4L), mRNAgi|34222182|ref|NM_145244.2 (SEQ ID NO: 14) SPP1 secreted phosphoprotein1 variant 1 gi|91206461|ref|NM_001040058.1 (SEQ ID NO: 15) variant 2gi|38146097|ref|NM_000582.2 (SEQ ID NO: 16) variant 3gi|91598938|ref|NM_001040060.1 (SEQ ID NO: 17) SOX9 Homo sapiens SRY(sex determining region Y)-box 9 (campomelic dysplasia, autosomalsex-reversal) (SOX9), mRNA gi|37704387|ref|NM_000346.2 (SEQ ID NO: 18)ACHE Homo sapiens acetylcholinesterase (Yt blood group) (ACHE), variantE4-E6, mRNA gi|88999567|ref|NM_000665.3 (SEQ ID NO: 19) variant E4-E5,mRNA gi|88999566|ref|NM_015831.2 (SEQ ID NO: 20) ADRB1 Homo sapiensadrenergic, beta-1-, receptor (ADRB1), mRNA gi|110349783|ref|NM_000684.2(SEQ ID NO: 21) HTRA2 Htra serine peptidase 2 v 1 gi:73747817, NM_013247(SEQ ID NO: 22) v 2 gi:73747818, NM_145074 (SEQ ID NO: 23) KEAP1Kelch-like ECH-associated protein 1 v 1 gi:45269144 NM_203500 (SEQ IDNO: 24) v 2 gi:45269143 NM_012289 (SEQ ID NO: 25) SHC1- Src homology 2domain containing transforming prot. 1 SHC v 1 gi: 194239661 NM_183001(SEQ ID NO: 26) v 2 gi: 194239660 NM_003029 (SEQ ID NO: 27) CAPNS1 Homosapiens calpain, small subunit 1 (CAPNS1), variant 1, mRNAgi|51599152|ref|NM_001749.2 (SEQ ID NO: 28) variant 2, mRNAgi|51599150|ref|NM_001003962.1 (SEQ ID NO: 29) PARP1 Homo sapiens poly(ADP-ribose) polymerase 1 (PARP1), mRNA gi|156523967|ref|NM_001618.3(SEQ ID NO: 30) AIF Homo sapiens apoptosis-inducing factor,mitochondrion-associated, 1 (AIFM1) variant 4, mRNAgi|195927003|ref|NM_001130846.1 (SEQ ID NO: 31) variant 5, mRNAgi|195927005|ref|NM_001130847.1 (SEQ ID NO: 32) NOS1 Homo sapiens nitricoxide synthase 1 (neuronal) (NOS1), mRNA gi|194239671|ref|NM_000620.2(SEQ ID NO: 33) NOS2A Homo sapiens nitric oxide synthase 2, inducible(NOS2), mRNA gi|206597519|ref|NM_000625.4 (SEQ ID NO: 34) XIAP Homosapiens X-linked inhibitor of apoptosis (XIAP), mRNAgi|32528298|ref|NM_001167.2| (SEQ ID NO: 35)

TABLE A2 Target genes for treatment of dry eye Gene Full name and HumanGene ID FAS CD95, TNF receptor superfamily, member 6 v 1.gi|23510419|ref|NM_000043.3| (SEQ ID NO: 36) v 3.gi|23510422|ref|NM_152872.1| (SEQ ID NO: 37) v 2.gi|23510420|ref|NM_152871.1| (SEQ ID NO: 38) v 4.gi|23510424|ref|NM_152873.1| (SEQ ID NO: 39) v 8.gi|23510426|ref|NM_152874.1| (SEQ ID NO: 40) v 5.gi|23510428|ref|NM_152875.1| (SEQ ID NO: 41) v 7.gi|23510433|ref|NM_152877.1| (SEQ ID NO: 42) v 6.gi|23510430|ref|NM_152876.1| (SEQ ID NO: 43) FAS TNF superfamily, member6 (FASL) ligand gi|4557328|ref|NM_000639.1| (SEQ ID NO: 44) p53 tumorprotein p53 gi8400737, NM_000546.2 (SEQ ID NO: 5) LRDD leucine-richrepeats and death domain containing gi|61742781|ref|NM_018494.3 (SEQ IDNO: 8) gi|61742783|ref|NM_145886.2 (SEQ ID NO: 9)gi|61742785|ref|NM_145887.2 (SEQ ID NO: 10) PARP1 Homo sapiens poly(ADP-ribose) polymerase 1 (PARP1), mRNA gi|156523967|ref|NM_001618.3(SEQ ID NO: 30) AIF Homo sapiens apoptosis-inducing factor,mitochondrion-associated, 1 (AIFM1) variant 4, mRNAgi|195927003|ref|NM_001130846.1 (SEQ ID NO: 31) variant 5, mRNAgi|195927005|ref|NM_001130847.1 (SEQ ID NO: 32) NOS1 Homo sapiens nitricoxide synthase 1 (neuronal) (NOS1), mRNA gi|194239671|ref|NM_000620.2(SEQ ID NO: 33) NOS2A Homo sapiens nitric oxide synthase 2, inducible(NOS2), mRNA gi|206597519|ref|NM_000625.4 (SEQ ID NO: 34) XIAP Homosapiens X-linked inhibitor of apoptosis (XIAP), mRNAgi|32528298|ref|NM_001167.2| (SEQ ID NO: 35) SHC1- Src homology 2 domaincontaining transforming prot. 1 SHC v 1 gi: 194239661 NM_183001 (SEQ IDNO: 26) v 2 gi: 194239660 NM_003029 (SEQ ID NO: 27)

TABLE A3 Target genes for treatment of DR, DME, AMD Gene Full name andHuman Gene ID LGALS3 lectin galactoside-binding soluble 3 v1 gi:115430222 NM_002306 (SEQ ID NO: 45) v2 gi: 115430224 NR_003225 (SEQ IDNO: 46) SLC2A1 solute carrier family 2 (facilitated glucosetransporter), member 1 gi|166795298|ref|NM_006516.1 (SEQ ID NO: 49)SLC2A2 solute carrier family 2 (facilitated glucose transporter), member2 gi|4557850|ref|NM_000340.1 (SEQ ID NO: 50) SLC2A3 Homo sapiens solutecarrier family 2 (facilitated glucose transporter), member 3 (GLUTS)gi|221136810|ref|NM_006931.1 (SEQ ID NO: 51) SLC5A1 solute carrierfamily 5 (sodium/glucose cotransporter), member 1gi|208973247|ref|NM_000343.2 (SEQ ID NO: 52) SORD sorbitol dehydrogenasemRNA gi|156627570|ref|NM_003104.3 (SEQ ID NO: 53) CTSD Homo sapienscathepsin D (CTSD) gi|23110949|ref|NM_001909.3 (SEQ ID NO: 47) CASP2caspase 2, apoptosis-related cysteine peptidasegi|39995058|ref|NM_032982.2 (SEQ ID NO: 1) gi|39995060|ref|NM_032983.2(SEQ ID NO: 2) RTP801 Homo sapiens DNA-damage-inducible transcript 4(DDIT4), mRNA gi|56676369|ref|NM_019058.2 (SEQ ID NO: 3) p53 tumorprotein p53 gi8400737, NM_000546.2 (SEQ ID NO: 5) TP53BP2 tumor proteinp53 binding protein, 2 gi|112799848|ref|NM_001031685.2 (SEQ ID NO: 6)gi|112799845|ref|NM_005426.2 (SEQ ID NO: 7) LRDD leucine-rich repeatsand death domain containing gi|61742781|ref|NM_018494.3 (SEQ ID NO: 8)gi|61742783|ref|NM_145886.2 (SEQ ID NO: 9) gi|61742785|ref|NM_145887.2(SEQ ID NO: 10) BNIP3 BCL2/adenovirus E1B 19 kDa interacting protein 3gi|7669480|ref|NM_004052.2 (SEQ ID NO: 12) RAC1 ras-related C3 botulinumtoxin substrate 1 (rho family, small GTP binding protein)(gi|38505164|ref|NM_198829.1) (SEQ ID NO: 13) AKR1B1 aldo-keto reductasefamily 1, member B1 (aldose reductase) gi|24497579|ref|NM_001628.2 (SEQID NO: 48) KEAP1 Kelch-like ECH-associated protein 1 v 1 gi:45269144NM_203500 (SEQ ID NO: 24) v 2 gi:45269143 NM_012289 (SEQ ID NO: 25)PARP1 Homo sapiens poly (ADP-ribose) polymerase 1 (PARP1), mRNAgi|156523967|ref|NM_001618.3 (SEQ ID NO: 30) AIF Homo sapiensapoptosis-inducing factor, mitochondrion-associated, 1 (AIFM1) variant4, mRNA gi|195927003|ref|NM_001130846.1 (SEQ ID NO: 31) variant 5, mRNAgi|195927005|ref|NM_001130847.1 (SEQ ID NO: 32) NOS1 Homo sapiens nitricoxide synthase 1 (neuronal) (NOS1), mRNA gi|194239671|ref|NM_000620.2(SEQ ID NO: 33) NOS2A Homo sapiens nitric oxide synthase 2, inducible(NOS2), mRNA gi|206597519|ref|NM_000625.4 (SEQ ID NO: 34) XIAP Homosapiens X-linked inhibitor of apoptosis (XIAP), mRNAgi|32528298|ref|NM_001167.21 (SEQ ID NO: 35) SHC1- Src homology 2 domaincontaining transforming prot. 1 SHC v 1 gi: 194239661 NM_183001 (SEQ IDNO: 26) v 2 gi: 194239660 NM_003029 (SEQ ID NO: 27)

TABLE A4 Examples of target genes for treatment of retinitis pigmentosa(RP) Gene Full name and Human Gene ID CASP1 Homo sapiens caspase 1,apoptosis-related cysteine peptidase (interleukin 1, beta, convertase)(CASP1), mRNA variant epsilon, gi|73622117|ref|NM_033295.2 (SEQ ID NO:54) variant alpha, gi|73622114|ref|NM_033292.2 (SEQ ID NO: 55) CASP3Homo sapiens caspase 3, apoptosis-related cysteine peptidase (CASP3),mRNA variant alpha, gi|73622121|ref|NM_004346.3 (SEQ ID NO: 56) variantbeta, gi|73622122|ref|NM_032991.2 (SEQ ID NO: 57) CASP12 Homo sapienscaspase 12 variant zeta (CASP12) mRNA, complete sequence; alternativelyspliced gi|20069120|gb|AF486846.1| (SEQ ID NO: 58) RTP801 Homo sapiensDNA-damage-inducible transcript 4 (DDIT4), mRNAgi|56676369|ref|NM_019058.2 (SEQ ID NO: 3) RTP801L Homo sapiensDNA-damage-inducible transcript 4-like (DDIT4L), mRNAgi|34222182|ref|NM_145244.2 (SEQ ID NO: 14) CAPNS1 Homo sapiens calpain,small subunit 1 (CAPNS1), variant 1, mRNA gi|51599152|ref|NM_001749.2(SEQ ID NO: 28) variant 2, mRNA gi|51599150|ref|NM_001003962.1 (SEQ IDNO: 29) PARP1 Homo sapiens poly (ADP-ribose) polymerase 1 (PARP1), mRNAgi|156523967|ref|NM_001618.3 (SEQ ID NO: 30) AIF Homo sapiensapoptosis-inducing factor, mitochondrion-associated, 1 (AIFM1) variant4, mRNA gi|195927003|ref|NM_001130846.1 (SEQ ID NO: 31) variant 5, mRNAgi|195927005|ref|NM_001130847.1 (SEQ ID NO: 32) NOS1 Homo sapiens nitricoxide synthase 1 (neuronal) (NOS1), mRNA gi|194239671|ref|NM_000620.2(SEQ ID NO: 33) NOS2A Homo sapiens nitric oxide synthase 2, inducible(NOS2), mRNA gi|206597519|ref|NM_000625.4 (SEQ ID NO: 34) XIAP Homosapiens X-linked inhibitor of apoptosis (XIAP), mRNAgi|32528298|ref|NM_001167.21 (SEQ ID NO: 35) SHC1- Src homology 2 domaincontaining transforming prot. 1 SHC v 1 gi: 194239661 NM_183001 (SEQ IDNO: 26) v 2 gi: 194239660 NM_003029 (SEQ ID NO: 27) “Variant” or “v”refer to transcript variant.

Tables A1-A4 provide the gi (GeneInfo identifier) and accession numbersfor an example of polynucleotide sequences of human mRNA to which theoligonucleotide inhibitors of the present invention are directed. (“v”refers to transcript variant)

Inhibition of the genes in Tables A1, A2, A3 and A4 is useful intreating, inter alia, glaucoma, dry eye, diabetic retinopathy (DR),diabetic macular edema (DME), age related macular degeneration (AMD) andretinitis pigmentosa (RP), respectively.

DEFINITIONS

For convenience certain terms employed in the specification, examplesand claims are described herein.

It is to be noted that, as used herein, the singular forms “a”, “an” and“the” include plural forms unless the content clearly dictatesotherwise.

Where aspects or embodiments of the invention are described in terms ofMarkush groups or other grouping of alternatives, those skilled in theart will recognize that the invention is also thereby described in termsof any individual member or subgroup of members of the group.

An “inhibitor” is a compound which is capable of reducing the expressionof a gene or the activity of the product of such gene to an extentsufficient to achieve a desired biological or physiological effect. Theterm “inhibitor” as used herein refers to one or more of anoligonucleotide inhibitor, including siRNA, antisense, shRNA, miRNA andribozymes. Inhibition may also be referred to as attenuation ofexpression of mRNA, down-regulation, or for RNA interference (RNAi),silencing. The inhibitors disclosed herein are chemically modified siRNAcompounds which incorporate modifications such as changes to the sugarmoiety and/or the base moiety and/or the linkages between nucleotides inthe oligonucleotide structure.

The term “inhibit” or “attenuate” as used herein refers to reducing theexpression of a gene, a variant or product thereof or the activity ofthe product of such gene to an extent sufficient to achieve a desiredbiological or physiological effect. Inhibition may be complete orpartial. For example “inhibition” of CASP2 gene means inhibition of thegene expression (transcription or translation) or polypeptide activityof one or more of the variants disclosed in any Table A1 or A3 or an SNP(single nucleotide polymorphism) or other variants thereof.

“Ocular tissue” referees to any tissue associated with structure of theeye and is intended to include, in a non-limiting manner, the sclera,the cornea, the choroid, the retina, the lacrimal gland, and the opticnerve.

“Ocular cell” refers to any cell associated with structures of the eyeand lacrimal apparatus and is intended to include, in a non-limitingmanner, retinal ganglion cell, a retinal pigment epithelial cell, acorneal cell, conjunctiva, anterior chamber, iris, ciliary process,retina, choroid and choroidal cells, trabecular meshwork, and the like.For example the trabecular meshwork includes the inner uveal meshwork,the corneoscleral meshwork and the juxtacanalicular tissue. The lacrimalgland includes the lacrimal gland per se, the inferior and superiorlacrimal puncta, the inferior and superior lacrimal canal, the lacrimalsac and the like.

As used herein, the terms “polynucleotide” and “nucleic acid” may beused interchangeably and refer to nucleotide sequences comprisingdeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). The termsshould also be understood to include, as equivalents, analogs of eitherRNA or DNA made from nucleotide analogs. Throughout this applicationmRNA sequences are set forth as representing the corresponding genes.The terms “mRNA polynucleotide sequence” and mRNA are usedinterchangeably.

“Oligonucleotide”, “oligoribonucleotide or “oligomer” refers to adeoxyribonucleotide or ribonucleotide sequence from about 2 to about 50nucleotides. Each DNA or RNA nucleotide of the sequence may beindependently natural or synthetic, and or modified or unmodified.Modifications include changes to the sugar moiety, the base moiety andor the linkages between nucleotides in the oligonucleotide. Thecompounds of the present invention encompass molecules comprisingdeoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides,modified ribonucleotides and combinations thereof.

The present invention provides methods and compositions for inhibitingexpression of a target gene in vivo. In general, the method includestopically administering oligoribonucleotides, in particular smallinterfering RNAs (i.e., siRNAs) or a nucleic acid material that canproduce siRNA in a cell, to target an mRNA of the genes set forth inTables A1-A4; in an amount sufficient to down-regulate expression of atarget gene by an RNA interference mechanism. In particular, the methodcan be used to inhibit expression of the gene for treatment of a subjectsuffering from an eye disorder or disease related to expression of thatgene in ocular tissue or cell. In accordance with the present invention,the siRNA molecules or inhibitors of the target gene are used as drugsto treat various ocular pathologies.

“Nucleotide” is meant to encompass deoxyribonucleotides andribonucleotides, which may be natural or synthetic, and or modified orunmodified. Modifications include changes and substitutions to the sugarmoiety, the base moiety and/or the internucleotide linkages.

As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which comprises a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide.

All analogs of, or modifications to, a nucleotide/oligonucleotide may beemployed with the present invention, provided that said analog ormodification does not substantially adversely affect the stability andfunction of the nucleotide/oligonucleotide. Acceptable modificationsinclude modifications of the sugar moiety, modifications of the basemoiety, modifications in the internucleotide linkages and combinationsthereof.

What is sometimes referred to in the present invention as an “abasicnucleotide” or “abasic nucleotide analog” is more properly referred toas a pseudo-nucleotide or an unconventional moiety. A nucleotide is amonomeric unit of nucleic acid, consisting of a ribose or deoxyribosesugar, a phosphate, and a base (adenine, guanine, thymine, or cytosinein DNA; adenine, guanine, uracil, or cytosine in RNA). A modifiednucleotide comprises a modification in one or more of the sugar,phosphate and or base. The abasic pseudo-nucleotide lacks a base, andthus is not strictly a nucleotide.

The term “capping moiety” as used herein includes abasic ribose moiety,abasic deoxyribose moiety, modifications abasic ribose and abasicdeoxyribose moieties including 2′ O alkyl modifications; inverted abasicribose and abasic deoxyribose moieties and modifications thereof;C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′OMenucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide;1-(3-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties. A 2′-O-methyl sugar modified ribonucleotide is also referredto as 2′-OMe sugar modified or 2′-OMe modified ribonucleotide

Certain preferred capping moieties are abasic ribose or abasicdeoxyribose moieties; inverted abasic ribose or abasic deoxyribosemoieties; C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA.

The term “unconventional moiety” as used herein refers to abasic ribosemoiety, an abasic deoxyribose moiety, a deoxyribonucleotide, a modifieddeoxyribonucleotide, a mirror nucleotide, a non-base pairing nucleotideanalog and a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond; bridged nucleic acids including LNA andethylene bridged nucleic acids.

Abasic deoxyribose moiety includes for example abasicdeoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribosemoiety includes inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic5′-phosphate.

In the context of the present invention, a “mirror” nucleotide (alsoreferred to as a spieglemer), is a nucleotide analog with reversechirality to the naturally occurring or commonly employed nucleotide,i.e., a mirror image of the naturally occurring or commonly employednucleotide. The mirror nucleotide is a ribonucleotide (L-RNA) or adeoxyribonucleotide (L-DNA) and may further comprise at least one sugaror base modification and/or a backbone modification, such as aphosphorothioate or phosphonate moiety. U.S. Pat. No. 6,602,858discloses nucleic acid catalysts comprising at least one L-nucleotidesubstitution. Mirror nucleotide includes for example L-DNA(L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror dT) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouracil-3′-phosphate (mirror dU)

Modified deoxyribonucleotide includes, for example 5′OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate.

Bridged nucleic acids include LNA (2′-O,4′-C-methylene bridged NucleicAcid adenosine 3′ monophosphate, 2′-O,4′-C-methylene bridged NucleicAcid 5-methyl-cytidine 3′ monophosphate, 2′-O,4′-C-methylene bridgedNucleic Acid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine)3′ monophosphate); and ENA (2′-O,4′-C-ethylene bridged Nucleic Acidadenosine 3′ monophosphate, 2′-O,4′-C-ethylene bridged Nucleic Acid5-methyl-cytidine 3′ monophosphate, 2′-O,4′-C-ethylene bridged NucleicAcid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine) 3′monophosphate).

In some embodiments of the present invention a preferred unconventionalmoiety is an abasic ribose moiety, an abasic deoxyribose moiety, adeoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to anadjacent nucleotide by a 2′-5′ internucleotide phosphate bond.

According to one aspect the present invention provides inhibitoryoligonucleotide compounds comprising unmodified and modifiednucleotides. The compound comprises at least one modified nucleotideselected from the group consisting of a sugar modification, a basemodification and an internucleotide linkage modification and may containDNA, and modified nucleotides such as LNA (locked nucleic acid)including ENA (ethylene-bridged nucleic acid; PNA (peptide nucleicacid); arabinoside; PACE (phosphonoacetate and derivatives thereof),mirror nucleotide, or nucleotides with a 6 carbon sugar.

In one embodiment the compound comprises a 2′ modification on the sugarmoiety of at least one ribonucleotide (“2′ sugar modification”). Incertain embodiments the compound comprises 2′O-alkyl or 2′-fluoro or2′O-allyl or any other 2′ sugar modification, optionally on alternatepositions.

Other stabilizing modifications are also possible (e.g. modifiednucleotides added to a 3′ or 5′ terminus of an oligomer). In someembodiments the backbone of the oligonucleotides is modified andcomprises phosphate-D-ribose entities but may also containthiophosphate-D-ribose entities, triester, thioate, 2′-5′ bridgedbackbone (also may be referred to as 5′-2′), PACE modifiedinternucleotide linkage or any other type of modification.

Other modifications include additions to the 5′ and/or 3′ termini of theoligonucleotides. Such terminal modifications may be lipids, peptides,sugars or other molecules.

Possible modifications to the sugar residue are manifold and include2′-O alkyl, locked nucleic acid (LNA), glycol nucleic acid (GNA),threose nucleic acid (TNA), arabinoside; altritol (ANA) and other6-membered sugars including morpholinos, and cyclohexinyls.

LNA compounds are disclosed in International Patent Publication Nos. WO00/47599, WO 99/14226, and WO 98/39352. Examples of siRNA compoundscomprising LNA nucleotides are disclosed in Elmen et al., (NAR 2005.33(1):439-447) and in International Patent Publication No. WO2004/083430. Six-membered ring nucleotide analogs are disclosed inAllart, et al (Nucleosides & Nucleotides, 1998, 17:1523-1526; andPerez-Perez, et al., 1996, Bioorg. and Medicinal Chem Letters6:1457-1460) Oligonucleotides comprising 6-membered ring nucleotideanalogs including hexitol and altritol nucleotide monomers are disclosedin International patent application publication No. WO 2006/047842.

Backbone modifications, such as ethyl (resulting in a phospho-ethyltriester); propyl (resulting in a phospho-propyl triester); and butyl(resulting in a phospho-butyl triester) are also possible. Otherbackbone modifications include polymer backbones, cyclic backbones,acyclic backbones, thiophosphate-D-ribose backbones, amidates, andphosphonoacetate derivatives. Certain structures include siRNA compoundshaving one or a plurality of 2′-5′ internucleotide linkages (bridges orbackbone).

The present invention also relates to compounds which down-regulateexpression of various genes, particularly to novel small interfering RNA(siRNA) compounds, and to the use of these novel siRNAs in the treatmentof various ocular diseases and medical conditions of the eye.

For each gene there is a separate list of 19-mer oligomer sequences,which are prioritized based on their score in the proprietary algorithmas the best sequences for targeting the human gene expression. 21- or23-mer siRNA sequences can be generated by 5′ and/or 3′ extension of the19-mer sequences disclosed herein. Such extension is preferablycomplementary to the corresponding mRNA sequence. Certain 23-meroligomers were devised by this method where the order of theprioritization is the order of the corresponding 19-mer. Tables B1-B36provide human sense and corresponding antisense oligonucleotides usefulin preparing siRNA. The abbreviations for cross species are: Ms: Mouse,Rb: Rabbit, Chmp: chimpanzee, Mnk: Monkey, Chn: chinchilla, GP:guinea-pig.

Ocular Diseases

Topical delivery of therapeutic oligonucleotide compounds is useful inthe treatment of a broad spectrum of eye diseases and disorders. Certainof the compounds of the invention are useful in treating patientssuffering from diseases and disorders in which neuroprotection of theoptic nerve would be of benefit, for example in:

1. open angle primary/secondary glaucoma2. multiple sclerosis (optic neuritis)3. central or brunch retinal vein occlusion4. ischemic optic neuropathy (in status epilepticus, HIV-1 infection)5. optic nerve injury6. tumors extending into the suprasellar region (above the sellaturcica)7. juxta chiasmal tumors (the visual loss associated with compression ofthe optic chiasm by pituitary tumors may be transient or permanent,possibly related to the extent of irreversible retrograde degenerationto the retinal ganglion cells.

8. Retinoblastoma Primary Open-Angle Glaucoma

The majority of the cases of glaucoma are the form known asprimary-open-angle glaucoma POAG, also called chronic open-angleglaucoma). POAG results from a build up of aqueous humor fluid withinthe anterior chamber of the eye resulting in intraocular pressure (IOP).Elevated IOP, which can be measured by a “tonometry” test, results fromfluid entering the eye and not enough fluid exiting the eye. Normally,fluid enters the eye by seeping out of the blood vessels in the ciliarybody. This fluid eventually makes its way past the crystalline lens,through the pupil (the central opening in the iris), and into theirido-corneal angle, the anatomical angle formed where the iris and thecornea come together. Then the fluid passes through the trabecularmeshwork in the angle and leaves the eye via the canal of Schlemm.

If excess fluid enters the eye, or if the trabecular meshwork “drain”gets clogged up (for instance, with debris or cells) so that not enoughfluid is leaving the eye, the pressure builds up in what is known as“open angle glaucoma.” Open angle glaucoma also can be caused when theposterior portion of the iris adheres to the anterior surface of thelens creating a “pupillary block”, and preventing intraocular fluid frompassing through the pupil into the anterior chamber.

If the angle between the iris and the cornea is too narrow or is evenclosed, then the fluid backs up, causing increased pressure in what isknown as “closed angle glaucoma.”

Untreated glaucoma eventually leads to optic atrophy and blindness.

Normal Tension Glaucoma

Intraocular eye pressure is normal (between 12-22 mmHg) in about 25-30%glaucoma cases in the US, a condition known as normal-tension glaucoma.(In Japan, the rates may be as high as 70%.) Other factors are presentthat cause optic nerve damage but do not affect IOP.

Closed-Angle Glaucoma

Closed-angle glaucoma (also called angle-closure glaucoma) isresponsible for 15% of all glaucoma cases. It is less common than POAGin the U.S., but it constitutes about half of the world's glaucoma casesbecause of its higher prevalence among Asians. The iris is pushedagainst the lens, sometimes sticking to it, closing off the drainageangle. This can occur very suddenly, resulting in an immediate rise inpressure. It often occurs in genetically susceptible people when thepupil shrinks suddenly. Closed-angle glaucoma can also be chronic andgradual, a less common condition.

Congenital Glaucoma

Congenital glaucoma, in which the eye's drainage canals fail to developcorrectly, is present from birth. It is very rare, occurring in about 1in 10,000 newborns. This may be an inherited condition and often can becorrected with microsurgery.

In one aspect the present invention provides a method of attenuatingexpression of a target ocular mRNA in the eye of a subject sufferingfrom glaucoma, comprising topically (non-invasively) administering tothe surface of the eye of the subject an effective amount of at leastone chemically modified siRNA and a pharmaceutically acceptable carrier.In certain embodiments the at least one ocular target mRNA is a productof a gene selected from a list in Table A1 set forth in SEQ ID NOS:1-35.In certain preferred embodiments the target ocular mRNA is a product ofa gene selected from CASP2, ASPP1, TP53BP2, BNIP3, RTP801L, ACHE, ADRB1and CAPNS1. In a currently preferred embodiment the siRNA is formulatedfor delivery as eye drops. In various embodiments the target ocular mRNAset forth in SEQ ID NOS:1-2.

In another aspect the present invention provides a method of treatingglaucoma in a subject in need of treatment, comprising topically(non-invasively) administering to the surface of the eye of the subjecta therapeutically effective amount of at least one chemically modifiedsiRNA which inhibits expression of a target gene in the eye of thesubject. In some embodiments the gene is a human gene selected from alist in Table A1, transcribed into mRNA set forth in any one of SEQ IDNOS:1-35. In certain preferred embodiments the gene is selected fromCASP2, ASPP1, TP53BP2, BNIP3, RTP801L, ACHE, ADRB1 and CAPNS1. In acurrently preferred embodiment the siRNA is formulated for delivery aseye drops. In various embodiments the target ocular mRNA set forth inSEQ ID NOS:1-2.

Optic Neuritis

Optic neuritis is an inflammation of the optic nerve that may affect thepart of the nerve and disc within the eyeball (papillitis) or theportion behind the eyeball (retrobulbar optic neuritis). Optic neuritismay be caused by any of the following: demyelinating diseases such asmultiple sclerosis or post infectious encephalomyelitis; systemic viralor bacterial infections including complications of inflammatory diseases(e.g., sinusitis, meningitis, tuberculosis, syphilis, chorioretinitis,orbital inflammation); nutritional and metabolic diseases (e.g.,diabetes, pernicious anemia, hyperthyroidism); Leber's Hereditary OpticNeuropathy, a rare form of inherited optic neuropathy which mainlyaffects young men; toxins (tobacco, methanol, quinine, arsenic,salicylates, lead); and trauma.

Optic Atrophy

Optic atrophy is a hereditary or acquired loss of vision disorder thatresults from the degeneration of the optic nerve and optic tract nervefibers. It may be acquired via occlusions of the central retinal vein orartery, arteriosclerotic changes, may be secondary to degenerativeretinal disease, may be a result of pressure against the optic nerve, ormay be related to metabolic diseases (e.g., diabetes), trauma, glaucoma,or toxicity (to alcohol, tobacco, or other poisons). Degeneration andatrophy of optic nerve fibers is irreversible, although intravenoussteroid injections have been seen to slow down the process in somecases.

Papilledema

A swelling of the optic disc (papilla), most commonly due to an increasein intracranial pressure (tumor induced), malignant hypertension, orthrombosis of the central retinal vein. The condition usually isbilateral, the nerve head is very elevated and swollen, and pupilresponse typically is normal. Vision is not affected initially and thereis no pain upon eye movement. Secondary optic nerve atrophy andpermanent vision loss can occur if the primary cause of the papilledemais left untreated.

Ischemic Optic Neuropathy (ION)

A severely blinding disease resulting from loss of the arterial bloodsupply to the optic nerve (usually in one eye), as a result of occlusivedisorders of the nutrient arteries. Optic neuropathy can be anterior,which causes a pale edema of the optic disc, or posterior, in which theoptic disc is not swollen and the abnormality occurs between the eyeballand the optic chiasm. Ischemic anterior optic neuropathy usually causesa loss of vision that may be sudden or occur over several days. Ischemicposterior optic neuropathy is uncommon, and the diagnosis dependslargely upon exclusion of other causes, chiefly stroke and brain tumor.

Other diseases and conditions include dry eye, diabetic retinopathy,diabetic macular edema and retinitis pigmentosa.

Dry Eye

Dry eye, also known as keratoconjunctivitis sicca or keratitis sicca, isa common problem usually resulting from a decrease in the production oftear film that lubricates the eyes. Most patients with dry eyeexperience discomfort, and no vision loss; although in severe cases, thecornea may become damaged or infected.

Dry eye is a multifactorial disease of the tears and ocular surface thatresults in symptoms of discomfort, visual disturbance, and tear filminstability with potential damage to the ocular surface. It isaccompanied by increased osmolarity of the tear film and inflammation ofthe ocular surface.

The lacrimal gland is a multilobular tissue composed of acinar, ductal,and myoepithelial cells. The acinar cells account for 80% of the cellspresent in the lacrimal gland and are the site for synthesis, storage,and secretion of proteins. Several of these proteins have antibacterial(lysozyme, lactoferrin) or growth factor (epidermal growth factor,transforming growth factor α, keratocyte growth factor) properties thatare crucial to the health of the ocular surface. The primary function ofthe ductal cells is to modify the primary fluid secreted by the acinarcells and to secrete water and electrolytes. The myoepithelial cellscontain multiple processes, which surround the basal area of the acinarand ductal cells, and are believed to contract and force fluid out ofthe ducts and onto the ocular surface.

Mechanisms of Lacrimal Gland Dysfunction

Apoptosis, hormonal imbalance, production of autoantibodies, alterationsin signaling molecules, neural dysfunction, and increased levels ofproinflammatory cytokines have been proposed as possible mediators oflacrimal gland insufficiency. One of the primary symptoms of SjögrensSyndrome is dry eye. Apoptosis of the acinar and ductal epithelial cellsof the lacrimal glands has been proposed as a possible mechanismresponsible for the impairment of secretory function (Manganelli andFietta, Semin Arthritis Rheum. 2003. 33(1):49-65). Without wishing to bebound by theory, apoptotic epithelial cell death may be due toactivation of several apoptotic pathways involving Fas (Apo-1/CD95),FasL (FasL/CD95L), Bax, caspases, perform, and granzyme B. Cytotoxic Tcells through the release of proteases, such as perform and granzyme B,or the interaction of FasL expressed by T cells with Fas on epithelialcells, can lead to apoptosis of the acinar cells.

The current treatment for dry eye is mainly local and symptomatic suchas: tear supplementation with lubricants; tear retention with therapiessuch as punctal occlusion, moisture chamber spectacles or contactlenses; tear stimulation for example by secretagogues; biological tearsubstitutes; anti-inflammatory therapy (Cyclosporine, Corticosteroids,Tetracyclines); and dietary essential fatty acids.

In one aspect the present invention provides a method of attenuatingexpression of a target ocular mRNA in the eye of a subject sufferingfrom dry-eye, comprising topically (non-invasively) administering to thesurface of the eye of the subject an effective amount of at least onechemically modified siRNA and a pharmaceutically acceptable carrier. Insome embodiments the target ocular mRNA is product of a human geneselected Table A2 set forth in SEQ ID NOS: 5, 8-10, 26-27, 30-44. Insome embodiments the target ocular mRNA is selected from FAS, FAS ligand(FASL), p53, LRDD, PARP1, AIF (apoptosis inducing factor), NOS1, NOS2A,XIAP and SHC1-SHC. In certain preferred embodiments the target ocularmRNA is set forth in any one of SEQ ID NOS:36-44. In a currentlypreferred embodiment the siRNA is formulated for delivery as eye drops.In various embodiments the subject is suffering from Sjögrens syndrome.In some embodiments the siRNA sense and antisense strands are selectedfrom sequences in Tables 16-17, set forth in SEQ ID NOS: 13225-15224.

In another aspect the present invention provides a method of treatingdry-eye in a subject in need of treatment, comprising topically(non-invasively) administering to the surface of the eye of the subjecta therapeutically effective amount of at least one chemically modifiedsiRNA which inhibits expression of a target gene in the eye of thesubject. In some embodiments the target gene is a human gene selectedfrom Table A2, whose mRNA is set forth in any one of SEQ ID NOS: 5,8-10, 26-27, or 30-44. In some embodiments the target gene is selectedfrom FAS, FAS ligand (FASL), p53, LRDD, PARP1, AIF (apoptosis inducingfactor), NOS1, NOS2A, XIAP and SHC1-SHC. In certain preferredembodiments the target gene mRNA is set forth in any one of SEQ IDNOS:36-44. In a currently preferred embodiment the siRNA is formulatedfor delivery as eye drops. In various embodiments the subject issuffering from Sjögrens syndrome. In some embodiments the siRNA senseand antisense strands are selected from sequences in Tables 16-17, setforth in SEQ ID NOS: 13225-15224.

Retinitis Pigmentosa

Retinitis pigmentosa (RP) represents a group of inherited disorderscharacterized by progressive peripheral vision loss and nyctalopia thatcan lead to central vision loss. There is currently no cure forretinitis pigmentosa, although the progression of the disease can beattenuated by dietary vitamin A supplementation.

Apoptosis in rod and cone photoreceptor cells is a critical process ofretinal degeneration in RP and represent a major cause of adultblindness.

In one aspect the present invention provides a method of attenuatingexpression of a target ocular mRNA in the eye of a subject sufferingfrom Retinitis pigmentosa (RP), comprising topically (non-invasively)administering to the surface of the eye of the subject an effectiveamount of at least one chemically modified siRNA and a pharmaceuticallyacceptable carrier. In some embodiments the target ocular mRNA isproduct of a gene selected from a gene listed in Table A4, the genetranscribed into mRNA set forth in any one of SEQ ID NOS: 3, 14, 26-35,54-57. In some embodiments the target ocular mRNA is product of a geneselected from the group consisting of CASP1, CASP3, CASP12, RTP801,RTP801L, Calpain 51, PARP1, AIF, NOS1, NOS2, XIAP and SHC1-SHC. In acurrently preferred embodiment the siRNA is formulated for delivery aseye drops. In certain preferred embodiments the siRNA targets mRNA setforth in SEQ ID NOS:56-57. In some embodiments the siRNA sense andantisense strands are selected from sequences in Table 11, set forth inSEQ ID NOS: 9517-10516.

In another aspect the present invention provides a method of treatingRetinitis pigmentosa (RP) in a subject in need of treatment, comprisingtopically (non-invasively) administering to the surface of the eye ofthe subject a therapeutically effective amount of at least onechemically modified siRNA which inhibits expression of a target gene inthe eye of the subject. In some embodiments the target gene is a humangene listed in Table A4, transcribed into mRNA set forth in any one ofSEQ ID NOS: 3, 14, 26-35, 54-57. In some embodiments the gene is a humangene selected from selected from the group consisting of CASP1, CASP3,CASP12, RTP801, RTP801L, Calpain 51, PARP1, AIF, NOS1, NOS2, XIAP andSHC1-SHC. In a currently preferred embodiment the siRNA is formulatedfor delivery as eye drops. In certain preferred embodiments the siRNAtargets and inhibits mRNA set forth in SEQ ID NOS:56-57. In someembodiments the siRNA sense and antisense strands are selected fromsequences in Table 11, set forth in SEQ ID NOS: 9517-10516.

Diabetic Retinopathy

Diabetic retinopathy is a complication of diabetes and a leading causeof blindness. It occurs when diabetes damages the tiny blood vesselsinside the retina. Diabetic retinopathy has four stages:

-   -   Mild Nonproliferative Retinopathy: microaneurysms in the        retina's blood vessels.    -   Moderate Nonproliferative Retinopathy. As the disease        progresses, some blood vessels that nourish the retina are        blocked.    -   Severe Nonproliferative Retinopathy. Many more blood vessels are        blocked, depriving several areas of the retina of a blood        supply, which is overcome by the growth of new blood vessels.    -   Proliferative Retinopathy. The new blood vessels grow along the        retina and along the surface of the vitreous gel. When the        vessels leak blood, severe vision loss and even blindness can        result.

During pregnancy, diabetic retinopathy may be a problem for women withdiabetes.

In one aspect the present invention provides a method of attenuatingexpression of a target ocular mRNA in the eye of a subject sufferingfrom diabetic retinopathy, comprising topically (non-invasively)administering to the surface of the eye of the subject an effectiveamount of at least one chemically modified siRNA and a pharmaceuticallyacceptable carrier. In some embodiments the target ocular mRNA isproduct of a human gene listed in Table A3 having mRNA set forth in anyone of SEQ ID NOS:1-2, 3, 5, 6-7, 8-10, 12, 13, 24-25, 26-27, 30-35,45-53. In a currently preferred embodiment the siRNA is formulated fordelivery as eye drops. In certain preferred embodiment the target ocularmRNA set forth in any one of SEQ ID NOS:48-53. In some embodiments thesiRNA sense and antisense strands are selected from sequences in Tables4, 28-32 set forth in SEQ ID NOS: 2669-3648 and 25575-29594.

In another aspect the present invention provides a method of treatingdiabetic retinopathy in a subject in need of treatment, comprisingtopically (non-invasively) administering to the surface eye of thesubject a therapeutically effective amount of at least one chemicallymodified siRNA which inhibits expression of a target gene in the eye ofthe subject. In some embodiments the target gene is a human gene listedin Table A3, having mRNA set forth in any one SEQ ID NOS:1-2, 3, 5, 6-7,8-10, 12, 13, 24-25, 26-27, 30-35, 45-53. In a currently preferredembodiment the siRNA is formulated for delivery as eye drops. In certainpreferred embodiment the target ocular mRNA set forth in any one of SEQID NOS:48-53. In some embodiments the siRNA sense and antisense strandsare selected from sequences in Tables 4, 28-32 set forth in SEQ ID NOS:2669-3648 and 25575-29594.

Without wishing to be bound to theory, blood vessels damaged fromdiabetic retinopathy can cause vision loss in two ways: Fragile,abnormal blood vessels can develop and leak blood into the center of theeye, blurring vision. This is proliferative retinopathy and is thefourth and most advanced stage of the disease. Fluid can leak into thecenter of the macula, resulting in blurred vision. This condition iscalled macular edema. It can occur at any stage of diabetic retinopathy,although it is more likely to occur as the disease progresses and isknown as diabetic macular edema (DME).

Age Related Macular Degeneration (AMD)

The most common cause of decreased best-corrected, vision in individualsover 65 years of age in the United States is the retinal disorder knownas age-related macular degeneration (AMD). The area of the eye affectedby AMD is the macula, a small area in the center of the retina, composedprimarily of photoreceptor cells. As AMD progresses, the disease ischaracterized by loss of sharp, central vision. So-called “dry” AMDaccounts for about 85%-90% of AMD patients and involves alterations ineye pigment distribution, loss of photoreceptors and diminished retinalfunction due to overall atrophy of cells. “Wet” AMD involvesproliferation of abnormal choroidal vessels leading to clots or scars inthe sub-retinal space. Thus, the onset of “wet” AMD occurs because ofthe formation of an abnormal choroidal neovascular network (choroidalneovascularization, CNV) beneath the neural retina. The newly formedblood vessels are excessively leaky. This leads to accumulation ofsubretinal fluid and blood leading to loss of visual acuity. Eventually,there is total loss of functional retina in the involved region, as alarge disciform scar involving choroids and retina forms. While dry AMDpatients may retain vision of decreased quality, wet AMD often resultsin blindness. (Hamdi & Kenney, Frontiers in Bioscience, e305-314, May2003).

In one aspect the present invention provides a method of attenuatingexpression of a target ocular mRNA in the eye of a subject sufferingfrom AMD or DME, comprising topically (non-invasively) administering tothe surface of the eye of the subject an effective amount of at leastone chemically modified siRNA and a pharmaceutically acceptable carrier.In some embodiments the target ocular mRNA is product of a human genelisted in Table A3, having mRNA set forth in any one of SEQ ID NOS:1-2,3, 5, 6-7, 8-10, 12, 13, 24-25, 26-27, 30-35, 45-53. In a currentlypreferred embodiment the siRNA is formulated for delivery as eye drops.In certain preferred embodiment the target ocular mRNA is product of ahuman CTSD, RTP801 and BNIP3.

In another aspect the present invention provides a method of treatingAMD or DME in a subject in need of treatment, comprising topically(non-invasively) administering to the surface of the eye of the subjecta therapeutically effective amount of at least one chemically modifiedsiRNA which inhibits expression of a target gene in the eye of thesubject. In some embodiments the target gene is a human gene listed inTable A3, having mRNA set forth in any one of SEQ ID NOS:1-2, 3, 5, 6-7,8-10, 12, 13, 24-25, 26-27, 30-35, 45-53. In a currently preferredembodiment the siRNA is formulated for delivery as eye drops. In certainpreferred embodiment the target human gene is a human CTSD, RTP801 andBNIP3.

Additional Ocular Conditions to be Treated by Compounds of the PresentInvention Viral and Bacterial Conditions

Viral and bacterial conditions relating to ocular tissues can be treatedby the compounds of the present invention. Conjunctivitis and othereyelid diseases or conditions can be treated, in particular byadministering according to the methods of the present inventionoligonucleotides such as siRNAs which target genes which are essentialfor replication and/or survival of the organisms which cause suchconditions.

Vision Loss Associated with Tumors

In another aspect the present invention provides method of treatingvision loss associated with a tumor in a subject in need thereof whichcomprises topically and non-invasively administering to the surface ofthe eye of the subject a therapeutically effective amount of at leastone chemically modified siRNA which inhibits expression of at least onegene associated with the tumor in the subject in an amount effective totreat the vision loss.

Tumors that cause vision loss, according to the present invention,include both malignant neoplasms (cancers) and benign tumors. Tumorsinclude tumors of any ocular tissue or any type of ocular cell,including, but not limited to, Choroidal tumors, Conjunctival tumors,Eyelid tumors, Infiltrative Intraocular tumors, Iris tumors, MetastaticOcular tumors, Optic Nerve tumors, Orbital tumors and Retinal tumors.More specifically, a non exhaustive list of tumors and cancers which thepresent invention aims to treat includes Choroidal Tumors such asChoroidal Hemangioma, Choroidal Melanoma, Choroidal Metastasis,Choroidal Nevus, Choroidal Osteoma, Ciliary Body Melanoma and Nevus ofOta; Conjunctival Tumors such as Conjunctival Kaposi's Sarcoma,Epibulbar Dermoid, Lymphoma of the Conjunctiva, Melanoma and PAM withAtypia, Pigmented Conjunctival Tumors, Pingueculum, Pterygium, SquamousCarcinoma and Intraepithelial Neoplasia of the Conjunctiva; EyelidTumors such as Basal Cell Carcinoma, Capillary Hemangioma, Hydrocystoma,Nevus at the Eyelid Margin, Seborrheic Keratosis, Malignant Melanoma ofthe Eyelid, Sebaceous Carcinoma of the Eyelid and Squamous Carcinoma ofthe Eyelid; Infiltrative Intraocular Tumors such as Chronic LymphocyticLeukemia, Infiltrative Choroidopathy and Intraocular Lymphoma; IrisTumors such as Anterior Uveal Metastasis, Iris Cysts, Iris Melanocytoma,Iris Melanoma and Pearl Cyst of the Iris; Metastatic Ocular Tumors suchas Metastatic Choroidal Melanoma; Optic Nerve Tumors such as ChoroidalMelanoma Affecting the Optic Nerve, Circumpapillary Metastasis withOptic Neuropathy, Optic Nerve Melanocytoma and Optic Nerve SheathMeningioma; Orbital Tumors such as Adenoid Cystic Carcinoma of theLacrimal Gland, Cavernous Hemangioma of the Orbit, Lymphangioma of theOrbit, Orbital Mucocele, Orbital Pseudotumor, Orbital Rhabdomyosarcoma,Periocular Hemangioma of Childhood and Sclerosing Orbital Pseudotumor;Retinal Tumors such as Retinal Pigment Epithelial (RPE) Hypertrophy,Retinal Pigment Epithelium (RPE) Tumors, Retinoblastoma and von HippelAngioma.

Pharmaceutical Compositions

While it may be possible for the oligonucleotide compounds of thepresent invention to be administered as the raw chemical, it ispreferable to administer them as a pharmaceutical composition.Accordingly the present invention provides a pharmaceutical compositioncomprising one or more of the compounds of the invention; and apharmaceutically acceptable carrier. This composition may comprise amixture of two or more different oligonucleotides/siRNAs.

The invention further provides a pharmaceutical composition comprisingat least one compound of the invention covalently or non-covalentlybound to one or more compounds of the invention in an amount effectiveto inhibit one or more genes as disclosed above; and a pharmaceuticallyacceptable carrier. The compound may be processed intracellularly byendogenous cellular complexes to produce one or moreoligoribonucleotides of the invention.

The invention further provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more of the compounds ofthe invention in an amount effective to down-regulate expression in of ahuman gene in an eye of a subject suffering from an eye disease ordisorder.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises:

-   -   providing one or more compounds of the invention; and    -   admixing said compound with a pharmaceutically acceptable        carrier.

The pharmaceutically acceptable carrier is preferably selected by onewith skill in the art for ophthalmological administration.

In various embodiments the pharmaceutical composition of the inventioncomprises at least one siRNA compound of the invention, or salt thereof,up to 99% by weight, mixed with a physiologically acceptable ophthalmiccarrier medium such as water, sodium chloride, buffer, saline (e.g.phosphate buffered saline (PBS)), mannitol, and the like, andcombinations thereof, to form an aqueous, sterile ophthalmic suspensionor solution.

The pharmaceutical composition further optionally comprises at least oneophthalmologically acceptable preservative, such as for examplebenzalkonium chloride. Further, the ophthalmic pharmaceuticalcomposition may include an ophthalmologically acceptable surfactant toassist in dissolving the siRNA.

Ophthalmic pharmaceutical composition of the invention may be preparedby dissolving or admixing one or more of the interfering RNA with anophthalmologically acceptable carrier, such as for example aphysiologically acceptable isotonic aqueous buffer.

In a preferred embodiment, the siRNA compound used in the preparation ofan ophthalmological composition is admixed with an ophthalmologicallyacceptable carrier in a pharmaceutically effective dose. In certainpreferred embodiments the ophthalmologically acceptable carrier is PBS.

In some embodiments the pharmaceutical ophthalmic compositions of theinvention further comprise additional pharmaceutically active agents ora combination of pharmaceutically active agent, such as non-steroidalanti-inflammatory drugs, corticosteroids, antibiotics, and the like.

Additionally, the invention provides a method of inhibiting theexpression of a gene of the present invention by at least 20% ascompared to a control comprising contacting an mRNA transcript of thegene of the present invention with one or more of the compounds of theinvention. In some embodiments an active siRNA compound inhibits geneexpression at a level of at least 20%, 30%, 40%, 50%, 60% or 70% ascompared to control. In certain preferred embodiments inhibition is at alevel of at least 75%, 80% or 90% as compared to control.

In one embodiment the oligoribonucleotide is inhibiting one or more ofthe genes of the present invention, whereby the inhibition is selectedfrom the group comprising inhibition of gene function, inhibition ofpolypeptide and inhibition of mRNA expression.

In one embodiment the compound inhibits a polypeptide, whereby theinhibition is selected from the group comprising inhibition of function(which may be examined by an enzymatic assay or a binding assay with aknown interactor of the native gene/polypeptide, inter alia), inhibitionof protein (which may be examined by Western blotting, ELISA orimmuno-precipitation, inter alia) and inhibition of mRNA expression(which may be examined by Northern blotting, quantitative RT-PCR,in-situ hybridization or microarray hybridization, inter alia).

Combination Therapy

The compounds of the present invention can be administered alone or incombination with another therapeutic agent useful in treating an eyedisorder or disease.

In some embodiments the pharmaceutical ophthalmic compositions of theinvention further comprise additional pharmaceutically active agents ora combination of pharmaceutically active agent, such as oligonucleotide,e.g. siRNA, non-steroidal anti-inflammatory drug, corticosteroid,antibiotic, and the like.

In one embodiment, the co-administration of two or more therapeuticagents achieves a synergistic effect, i.e., a therapeutic affect that isgreater than the sum of the therapeutic effects of the individualcomponents of the combination. In another embodiment, theco-administration of two or more therapeutic agents achieves an additiveeffect.

The active ingredients that comprise a combination therapy may beadministered together via a single dosage form or by separateadministration of each active agent. In certain embodiments, the firstand second therapeutic agents are administered in a single dosage form.The agents may be formulated into a single solution for topicaladministration. Alternatively, the first therapeutic agent and thesecond therapeutic agents may be administered as separate compositions.The first active agent may be administered at the same time as thesecond active agent or the first active agent may be administeredintermittently with the second active agent. The length of time betweenadministration of the first and second therapeutic agent may be adjustedto achieve the desired therapeutic effect. For example, the secondtherapeutic agent may be administered only a few minutes (e.g., 1, 2, 5,10, 30, or 60 min) or several hours (e.g., 2, 4, 6, 10, 12, 24, or 36hr) after administration of the first therapeutic agent. In certainembodiments, it may be advantageous to administer more than one dosageof one of the therapeutic agents between administrations of the secondtherapeutic agent. For example, the second therapeutic agent may beadministered at 2 hours and then again at 10 hours followingadministration of the first therapeutic agent. Alternatively, it may beadvantageous to administer more than one dosage of the first therapeuticagent between administrations of the second therapeutic agent. Incertain embodiments it is preferred that the therapeutic effects of eachactive ingredient overlap for at least a portion of the duration of eachtherapeutic agent so that the overall therapeutic effect of thecombination therapy is attributable in part to the combined orsynergistic effects of the combination therapy.

Delivery

The siRNA molecules as disclosed herein are delivered to the targettissue of the eye by direct application of the molecules prepared with acarrier or a diluent.

The term “naked siRNA” refers to siRNA molecules that are free from anydelivery vehicle or formulation that acts to assist, promote orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. For example, siRNA in PBS or an acceptable ophthalmologicalformulation is “naked siRNA”. In certain embodiments of the inventionthe siRNA is delivered as naked siRNA. For certain applications, aformulation that increases the residence time of the siRNA in the eye ornasal passage may be desired, for example, by addition of a polymer orviscosity enhancing agent.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, (see, forexample, Shen et al FEBS Let. 2003, 539:111-114; Xia et al., Nat.Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9:210-216; Sorensen et al., J. Mol. Biol. 2003. 327: 761-766; Lewis etal., Nat. Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11:2717-2724).

The pharmaceutically acceptable carriers, solvents, diluents,excipients, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention and they include liposomes and microspheres. Examples ofdelivery systems useful in the present invention include U.S. Pat. Nos.5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many othersuch implants, delivery systems, and modules are well known to thoseskilled in the art.

A review of the considerations to be taken into account in thepreparation of a pharmaceutical composition for topical ocular deliveryor intranasal delivery, can be found in Bar-Ilan and Neumann, inTextbook of Ocular Pharmacology, Zimmerman et al eds., Lippencott-Raven1997.

Topical routes of administration are preferably employed for providingthe subject with an effective dosage of the therapeutic siRNA compounds.Dosage forms may include dispersions, suspensions, solutions, ointmentsand the like. In certain preferred embodiments the pharmaceuticalcomposition of the invention comprising at least one siRNA is deliveredas eye drops. In another embodiment the pharmaceutical composition ofthe invention comprising at least one siRNA is delivered as a spray ormist. For example U.S. Pat. No. 4,052,985 discloses ophthalmic sprayapplicators.

In certain embodiments of the present invention the siRNA reaches itstarget cell locally, for example direct contact or by diffusion throughcells, tissue or intracellular fluid. The siRNAs or pharmaceuticalcompositions of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the disease to be treated,scheduling of administration, patient age, sex, body weight and otherfactors known to medical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited to animproved course of disease, more rapid recovery, improvement ofsymptoms, elimination of symptoms and other indicators as are selectedas appropriate measures by those skilled in the art. The siRNA of theinvention can be administered in a single dose or in multiple doses.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg/kg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a single doseor multiple dose regimen. In certain embodiments the siRNA compounds areformulated for topical application to the eye as eye drops and compriseabout 5 μg/μl to about 60 μg/μl by volume of the composition, about 6.6μg/μl by volume of the composition, about 25 μg/μl by volume of thecomposition, about 33.3 μg/μl by volume of the composition, about 50μg/μl by volume of the composition.

The pH of the formulation is about pH 5 to about pH 8, or about pH 5 toabout pH 7, or from about pH 5 to about pH 6. In certain embodiments thepH is about Ph 5.9, about pH 6.15, about pH 6.25, about pH 6.3, about pH6.5, about pH 7.25. The compounds of the present invention can beadministered topically to the surface of the eye. It should be notedthat the compound is preferably applied as the compound or aspharmaceutically acceptable salt active ingredient in combination withpharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and or vehicles. As disclosed herein the preferred method ofdelivery is topical application of an ophthalmic composition to the eye.

Liquid forms may be prepared for drops or spray. The liquid compositionsinclude aqueous solutions, with and without organic co-solvents, aqueousor oil suspensions, emulsions with edible oils, as well as similarpharmaceutical vehicles.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration to the eye, as by, forexample, a spray or drops, and topically, as by ointments, or drops.

Methods of Treatment

In one aspect, the present invention relates to a method for thetreatment of a subject in need of treatment for an eye disease ordisorder associated with expression of a gene listed in Tables A1-A4 inthe eye of the subject, comprising topically and non-invasivelyadministering to the subject an amount of a chemically modified siRNAwhich inhibits expression of at least one of the genes. In certainpreferred embodiments more than one siRNA compound to one or more thanone gene target is administered.

In preferred embodiments the subject being treated is a warm-bloodedanimal and, in particular, mammals including human.

The methods of the invention comprise topically and non-invasivelyadministering to the eye of the subject one or more inhibitory compoundswhich down-regulate expression of the genes of Tables A1-A4; and inparticular siRNA in a therapeutically effective dose so as to therebytreat the subject.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down, attenuate the related eye disorder as listed above. Thosein need of treatment include those already experiencing the disease orcondition, those prone to having the disease or condition, and those inwhich the disease or condition is to be prevented. The compounds of theinvention may be administered before, during or subsequent to the onsetof the eye disease or condition or symptoms associated therewith. Incases where treatment is for the purpose of prevention, then the presentinvention relates to a method for delaying the onset of or averting thedevelopment of the disease or disorder.

Ocular disorders include Acute Zonal Occult Outer Retinopathy, AdieSyndrome, Age Related Macular Degeneration, Amblyopia, Aniridia,Anisocoria, Anophthalmos, Aphakia, Blepharitis, Blepharoptosis,Blepharospasm, Blindness, Cataract, Chalazion, Chorioretinitis,Choroideremia, Coloboma, Conjunctival Diseases, Conjunctivitis, CornealDiseases, Corneal Dystrophies, Corneal Edema, Corneal Ulcer, DiabeticMacular Edema, Diabetic Retinopathy, Diplopia, Distichiasis, Dry EyeSyndromes, Duane Retraction Syndrome, Ectropion, Endophthalmitis,Entropion, Esotropia, Exfoliation Syndrome, Exotropia, EyeAbnormalities, Eye Neoplasms, General Fibrosis Syndrome, Glaucomas,Gyrate Atrophy, Hemianopsia, Hermanski-Pudlak Syndrome, Hordeolum,Horner Syndrome, Hyperopia, Hyphema, Iritis, Kearns-Sayer Syndrome,Keratitis, Keratoconus, Lacrimal Apparatus Diseases, Lacrimal DuctObstruction, Lens Diseases, Macular Degeneration, Nystagmus, Pathologic,Ocular Motility Disorders, Oculomotor Nerve Diseases, Ophthalmoplegia,Optic Atrophies, Hereditary, Optic Nerve Diseases, Optic Neuritis,Ischemic Optic Neuropathy, Orbital Cellulitis, Papilledema, Presbyopia,Pterygium, Pupil Disorders, Refractive Errors, Retinal Detachment,Retinal Diseases, Retinal Vein Occlusion, Retinal Blastoma, RetinitisPigmentosa, Retinopathy of Prematurity, Retinoschisis, Scleritis,Scotoma, Strabismus, Sjögrens Syndrome, Thygeson's Superficial PunctateKeratitis, Trachoma, Uveitis.

Oligonucleotides

The oligonucleotides useful in the methods disclosed herein arepreferably double stranded oligonucleotides and siRNA compounds andinclude unmodified and chemically and/or structurally modifiedcompounds.

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; see for example Ui-Tei et al., J BiomedBiotechnol. 2006; 65052; Chalk et al., BBRC. 2004, 319(1):264-74; Sioud& Leirdal, Met. Mol Biol. 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48. For examplesof the use and production of modified siRNA see for example Braasch etal., Biochem. 2003, 42(26):7967-75; Chiu et al., RNA. 2003,9(9):1034-48; PCT Publication Nos. WO 2004/015107 and WO 02/44321 andU.S. Pat. Nos. 5,898,031 and 6,107,094.

The present invention provides double-stranded oligonucleotides (e.g.siRNAs), which down-regulate the expression of a desired gene. A siRNAof the invention is a duplex oligoribonucleotide in which the sensestrand is derived from the mRNA sequence of the desired gene, and theantisense strand is complementary to the sense strand. In general, somedeviation from the target mRNA sequence is tolerated withoutcompromising the siRNA activity (see e.g. Czauderna et al., NAR. 2003,31(11):2705-2716). An siRNA of the invention inhibits gene expression ona post-transcriptional level with or without destroying the mRNA.Without being bound by theory, siRNA may target the mRNA for specificcleavage and degradation and/or may inhibit translation from thetargeted message.

In some embodiments the siRNA is blunt ended, i.e. Z and Z′ are absent,on one or both ends. More specifically, the siRNA may be blunt ended onthe end defined by the 5′-terminus of the first strand and the3′-terminus of the second strand, and/or the end defined by the3′-terminus of the first strand and the 5′-terminus of the secondstrand.

In other embodiments at least one of the two strands may have anoverhang of at least one nucleotide at the 5′-terminus; the overhang mayconsist of at least one deoxyribonucleotide. At least one of the strandsmay also optionally have an overhang of at least one nucleotide at the3′-terminus. The overhang may consist of from about 1 to about 5nucleotides.

The length of RNA duplex is from about 18 to about 40 ribonucleotides,preferably 19, 21 or 23 ribonucleotides. Further, the length of eachstrand may independently have a length selected from the groupconsisting of about 15 to about 40 bases, preferably 18 to 23 bases andmore preferably 19, 21 or 23 ribonucleotides. In some embodiments a 20or 22-mer molecule may be contemplated.

In certain embodiments the complementarity between said first strand andthe target nucleic acid is perfect (100%). In some embodiments, thestrands are substantially complementary, i.e. having one, two or up tothree mismatches between said first strand and the target nucleic acid.Substantially complementary refers to complementarity of greater thanabout 84%, to another sequence. For example in a duplex regionconsisting of 19 base pairs one mismatch results in 94.7%complementarity, two mismatches results in about 89.5% complementarityand 3 mismatches results in about 84.2% complementarity, rendering theduplex region substantially complementary. Accordingly substantiallyidentical refers to identity of greater than about 84%, to anothersequence.

The first strand and the second strand may be linked by a loopstructure, which may be comprised of a non-nucleic acid polymer such as,inter alia, polyethylene glycol. Alternatively, the loop structure maybe comprised of a nucleic acid, including modified and non-modifiedribonucleotides and modified and non-modified deoxyribonucleotides.

Further, the 5′-terminus of the first strand of the siRNA may be linkedto the 3′-terminus of the second strand, or the 3′-terminus of the firststrand may be linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 2-100 nucleobases, preferably about 2 to about 30 nucleobases.

In preferred embodiments of the compounds of the invention havingalternating ribonucleotides modified in at least one of the antisenseand the sense strands of the compound, for 19-mer and 23-mer oligomersthe ribonucleotides at the 5′ and 3′ termini of the antisense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the sense strand are unmodified in their sugar residues.For 21-mer oligomers the ribonucleotides at the 5′ and 3′ termini of thesense strand are modified in their sugar residues, and theribonucleotides at the 5′ and 3′ termini of the antisense strand areunmodified in their sugar residues, or may have an optional additionalmodification at the 3′ terminus. As mentioned above, it is preferredthat the middle nucleotide of the antisense strand is unmodified.

Additionally, the invention provides siRNA comprising a double strandednucleic acid molecule wherein 1, 2, or 3 of the nucleotides in onestrand or both strands are substituted thereby providing at least onebase pair mismatch. The substituted nucleotides in each strand arepreferably in the terminal region of one strand or both strands.

According to one preferred embodiment of the invention, the antisenseand the sense strands of the oligonucleotide/siRNA are phosphorylatedonly at the 3′-terminus and not at the 5′-terminus. According to anotherpreferred embodiment of the invention, the antisense and the sensestrands are non-phosphorylated. According to yet another preferredembodiment of the invention, the 5′ most ribonucleotide in the sensestrand is modified to abolish any possibility of in vivo5′-phosphorylation.

Any siRNA sequence disclosed herein can be prepared having any of themodifications/structures disclosed herein. The combination of sequenceplus structure is novel and can be used in the treatment of theconditions disclosed herein.

siRNA Structures

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; (see for example Ui-Tei et al., J Biomed Biotech.2006; 2006: 65052; Chalk et al., BBRC. 2004, 319(1): 264-74; Sioud &Leirdal, Met. Mol Biol.; 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48).

For examples of the use of, and production of, modified siRNA see, forexample, Braasch et al., Biochem. 2003, 42(26):7967-75; Chiu et al.,RNA, 2003, 9(9):1034-48; PCT publications WO 2004/015107 (atugen AG) andWO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094,teach chemically modified oligomers. US Patent Publication Nos.2005/0080246 and 2005/0042647 relate to oligomeric compounds having analternating motif and dsRNA compounds having chemically modifiedinternucleoside linkages, respectively.

Other modifications have been disclosed. The inclusion of a 5′-phosphatemoiety was shown to enhance activity of siRNAs in Drosophila embryos(Boutla, et al., Curr. Biol. 2001, 11:1776-1780) and is required forsiRNA function in human HeLa cells (Schwarz et al., Mol. Cell, 2002,10:537-48). Amarzguioui et al., (NAR, 2003, 31(2):589-95) showed thatsiRNA activity depended on the positioning of the 2′-O-methyl (2′-OMe)modifications. Holen et al (NAR. 2003, 31(9):2401-07) report that ansiRNA having small numbers of 2′-OMe modified nucleosides gave goodactivity compared to wild type but that the activity decreased as thenumbers of 2′-OMe modified nucleosides was increased. Chiu and Rana(RNA. 2003, 9:1034-48) teach that incorporation of 2′-OMe modifiednucleosides in the sense or antisense strand (fully modified strands)severely reduced siRNA activity relative to unmodified siRNA. Theplacement of a 2′-OMe group at the 5′-terminus on the antisense strandwas reported to severely limit activity whereas placement at the3′-terminus of the antisense and at both termini of the sense strand wastolerated (Czauderna et al., NAR. 2003, 31(11):2705-16; WO 2004/015107).The molecules of the present invention offer an advantage in that theyare non-toxic and may be formulated as pharmaceutical compositions fortreatment of various diseases.

The nucleotides can be selected from naturally occurring or syntheticmodified bases. Naturally occurring bases include adenine, guanine,cytosine, thymine and uracil. Modified bases of nucleotides includeinosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl andother alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine andother substituted guanines, other aza and deaza adenines, other aza anddeaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.Molecules comprising one or more abasic moiety (unconventional orpseudonucleotide) are encompassed by the present invention, as well asmolecules comprising alternating RNA and DNA nucleotides.

In addition, analogues of polynucleotides can be prepared wherein thestructure of one or more nucleotide is fundamentally altered and bettersuited as therapeutic or experimental reagents. An example of anucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogs have been shown to be resistant to enzymatic degradation andto have extended lives in vivo and in vitro.

Possible modifications to the sugar residue are manifold and include2′-O alkyl, locked nucleic acid (LNA), glycol nucleic acid (GNA),threose nucleic acid (TNA), arabinoside, altritol (ANA) and other,6-membered sugars including morpholinos, and cyclohexinyls. Further,said molecules may additionally contain modifications on the sugar, suchas 2′ alkyl, 2′ fluoro, 2′O allyl, 2′ amine and 2′ alkoxy. Additionalsugar modifications are discussed herein.

LNA compounds are disclosed in International Patent Publication Nos. WO00/47599, WO 99/14226, and WO 98/39352. Examples of siRNA compoundscomprising LNA nucleotides are disclosed in Elmen et al., (NAR 2005.33(1):439-447) and in PCT Patent Publication No. WO 2004/083430.

The compounds of the present invention can be synthesized using one ormore inverted nucleotides, for example inverted thymidine or invertedadenine (for example see Takei, et al., 2002. JBC 277(26):23800-06).

Backbone modifications, such as ethyl (resulting in a phospho-ethyltriester); propyl (resulting in a phospho-propyl triester); and butyl(resulting in a phospho-butyl triester) are also possible.

Other backbone modifications include polymer backbones, cyclicbackbones, acyclic backbones, thiophosphate-D-ribose backbones,amidates, and phosphonoacetate derivatives. Certain structures includesiRNA compounds having one or a plurality of 2′-5′ internucleotidelinkages (bridges or backbone).

Further, the inhibitory nucleic acid molecules of the present inventionmay comprise one or more gaps and/or one or more nicks and/or one ormore mismatches. Without wishing to be bound by theory, gaps, nicks andmismatches have the advantage of partially destabilizing the nucleicacid/siRNA, so that it may be more easily processed by endogenouscellular machinery such as DICER, DROSHA or RISC into its inhibitorycomponents.

The molecules of the present invention may comprise siRNAs, syntheticsiRNAs, shRNAs and synthetic shRNAs, in addition to other nucleic acidsequences or molecules which encode such molecules or other inhibitorynucleotide molecules.

The compounds of the present invention may further comprise an endmodification. A biotin group may be attached to either the most 5′ orthe most 3′ nucleotide of the first and/or second strand or to bothends. In a more preferred embodiment the biotin group is coupled to apolypeptide or a protein. It is also within the scope of the presentinvention that the polypeptide or protein is attached through any of theother aforementioned modifications.

The various end modifications as disclosed herein are preferably locatedat the ribose moiety of a nucleotide of the nucleic acid as disclosedherein. More particularly, the end modification may be attached to orreplace any of the OH-groups of the ribose moiety, including but notlimited to the 2′OH, 3′OH and 5′OH position, provided that thenucleotide thus modified is a terminal nucleotide. Inverted abasic orabasic are nucleotides, either deoxyribonucleotides or ribonucleotideswhich do not have a nucleobase moiety. This kind of compound is, interalia, described in Sternberger, et al., (Antisense Nucleic Acid DrugDev, 2002.12, 131-43).

In the context of the present invention, a gap in a nucleic acid refersto the absence of one or more internal nucleotides in one strand, whilea nick in a nucleic acid refers to the absence of an internucleotidelinkage between two adjacent nucleotides in one strand. Any of themolecules of the present invention may contain one or more gaps and/orone or more nicks. Further provided by the present invention is an siRNAencoded by any of the molecules disclosed herein, a vector encoding anyof the molecules disclosed herein, and a pharmaceutical compositioncomprising any of the molecules disclosed herein or the vectors encodingthem; and a pharmaceutically acceptable carrier.

Particular molecules to be administered according to the methods of thepresent invention are disclosed below under the heading “structuralmotifs”. For the sake of clarity, any of these molecules can beadministered according to any of the methods of the present invention.

Structural Motifs

As disclosed herein the siRNA compounds that are chemically and orstructurally modified according to one of the following modificationsset forth in Structures below or as tandem siRNA or RNAstar (see below)are useful in the methods of the present invention. Tables 1-36 providesense and antisense oligonucleotide pairs, set forth in SEQ IDNOS:59-33596, useful in preparing corresponding siRNA compounds.

In one aspect the present invention provides a compound set forth asStructure (A):

(A) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)wherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide and a modified deoxyribonucleotide;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of x and y is an integer between 18 and 40;wherein each of Z and Z′ may be present or absent, but if present is 1-5consecutive nucleotides covalently attached at the 3′ terminus of thestrand in which it is present; andand wherein the sequence of (N)_(x) comprises an antisense sequencesubstantially complementary to about 18 to about 40 consecutiveribonucleotides in the mRNA of a gene expressed in the retina andassociated with an ocular disease or disorder. In some embodiments themRNA is set forth in any one of SEQ ID NOS:1-58.

In certain embodiments the present invention provides a compound havingstructure B

(B) 5′ (N)x 3′ antisense strand 3′ (N′)y 5′ sense strandwherein each of (N)_(x) and (N′)_(y) is an oligomer in which eachconsecutive N or N′ is an unmodified ribonucleotide or a modifiedribonucleotide joined to the next N or N′ by a covalent bond;wherein each of x and y=19, 21 or 23 and (N)_(x) and (N′)_(y) are fullycomplementarywherein alternating ribonucleotides in each of (N)_(x) and (N′)_(y)comprise 2′-OMe sugar modified ribonucleotides;wherein the sequence of (N′)_(y) is a sequence complementary to (N)x;and wherein the sequence of (N)_(x) comprises an antisense sequencesubstantially complementary to about 18 to about 40 consecutiveribonucleotides in the mRNA set forth in any one of SEQ ID NOS:1-58.

In some embodiments each of (N)_(x) and (N′)_(y) is independentlyphosphorylated or non-phosphorylated at the 3′ and 5′ termini.

In certain embodiments of the invention, alternating ribonucleotides aremodified in both the antisense and the sense strands of the compound.

In certain embodiments wherein each of x and y=19 or 23, each N at the5′ and 3′ termini of (N)_(x) is modified; and

each N′ at the 5′ and 3′ termini of (N′)_(y) is unmodified.

In certain embodiments wherein each of x and y=21, each N at the 5′ and3′ termini of (N)_(x) is unmodified; and each N′ at the 5′ and 3′termini of (N′)_(y) is modified.

In particular embodiments, when x and y=19, the siRNA consists of a2′OMe sugar modified ribonucleotides on the first, third, fifth,seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth andnineteenth nucleotide of the antisense strand (N)_(x), and 2′-OMe sugarmodified ribonucleotides in the second, fourth, sixth, eighth, tenth,twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sensestrand (N′)_(y). In various embodiments these particular siRNA compoundsare blunt ended at both termini.

In some embodiments, the present invention provides a compound havingStructure (C):

(C) 5′ (N)x-Z 3′ antisense strand 3′ Z′-(N′)y 5′ sense strandwherein each of N and N′ is a nucleotide independently selected from anunmodified ribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide and a modified deoxyribonucleotide;wherein each of (N)x and (N′)y is an oligomer in which each consecutivenucleotide is joined to the next nucleotide by a covalent bond and eachof x and y is an integer between 18 and 40;wherein in (N)x the nucleotides are unmodified or (N)x comprisesalternating 2′ O Me sugar modified ribonucleotides and unmodifiedribonucleotides; and wherein the ribonucleotide located at the middleposition of (N)x being 2′OMe sugar modified or unmodified, preferablyunmodified;wherein (N′)y comprises unmodified ribonucleotides further comprisingone modified nucleotide at a terminal or penultimate position, whereinthe modified nucleotide is selected from the group consisting of amirror nucleotide, a bicyclic nucleotide, a 2′-sugar modifiednucleotide, an altritol nucleotide, or a nucleotide joined to anadjacent nucleotide by an internucleotide linkage selected from a 2′-5′phosphodiester bond, a P-alkoxy linkage or a PACE linkage;wherein if more than one nucleotide is modified in (N′)y, the modifiednucleotides may be consecutive;wherein each of Z and Z′ may be present or absent, but if present is 1-5deoxyribonucleotides covalently attached at the 3′ terminus of anyoligomer to which it is attached;wherein the sequence of (N′)_(y) comprises a sequence substantiallycomplementary to (N)x; and wherein the sequence of (N)_(x) comprises anantisense sequence substantially complementary to about 18 to about 40consecutive ribonucleotides in the mRNA set forth in any one of SEQ IDNOS:1-58.

In particular embodiments, x=y=19 and in (N)x each modifiedribonucleotide is a 2′-OMe sugar modified and the ribonucleotide locatedat the middle of (N)x is unmodified. Accordingly, in a compound whereinx=19, (N)x comprises 2′-O-methyl sugar modified ribonucleotides atpositions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In other embodiments,(N)x comprises 2′-OMe sugar modified ribonucleotides at positions 2, 4,6, 8, 11, 13, 15, 17 and 19 and may further comprise at least one abasicor inverted abasic pseudo-nucleotide for example in position 5. In otherembodiments, (N)x comprises 2′-OMe modified ribonucleotides at positions2, 4, 8, 11, 13, 15, 17 and 19 and may further comprise at least oneabasic or inverted abasic pseudo-nucleotide for example in position 6.In other embodiments, (N)x comprises 2′-OMe modified ribonucleotides atpositions 2, 4, 6, 8, 11, 13, 17 and 19 and may further comprise atleast one abasic or inverted abasic pseudo-nucleotide for example inposition 15. In other embodiments, (N)x comprises 2′-OMe modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and mayfurther comprise at least one abasic or inverted abasicpseudo-nucleotide for example in position 14. In other embodiments, (N)xcomprises 2′-OMe modified ribonucleotides at positions 1, 2, 3, 7, 9,11, 13, 15, 17 and 19 and may further comprise at least one abasic orinverted abasic pseudo-nucleotide for example in position 5. In otherembodiments, (N)x comprises 2′-OMe modified ribonucleotides at positions1, 2, 3, 5, 7, 9, 11, 13, 15, 17 and 19 and may further comprise atleast one abasic or inverted abasic pseudo-nucleotide for example inposition 6. In other embodiments, (N)x comprises 2′-OMe modifiedribonucleotides at positions 1, 2, 3, 5, 7, 9, 11, 13, 17 and 19 and mayfurther comprise at least one abasic or inverted abasicpseudo-nucleotide for example in position 15. In other embodiments, (N)xcomprises 2′-OMe modified ribonucleotides at positions 1, 2, 3, 5, 7, 9,11, 13, 15, 17 and 19 and may further comprise at least one abasic orinverted abasic pseudo-nucleotide for example in position 14. In otherembodiments, (N)x comprises 2′-OMe modified ribonucleotides at positions2, 4, 6, 7, 9, 11, 13, 15, 17 and 19 and may further comprise at leastone abasic or inverted abasic pseudo-nucleotide for example in position5. In other embodiments, (N)x comprises 2′-OMe modified ribonucleotidesat positions 1, 2, 4, 6, 7, 9, 11, 13, 15, 17 and 19 and may furthercomprise at least one abasic or inverted abasic pseudo-nucleotide forexample in position 5. In other embodiments, (N)x comprises 2′-OMemodified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 14, 16, 17 and19 and may further comprise at least one abasic or inverted abasicpseudo-nucleotide for example in position 15. In other embodiments, (N)xcomprises 2′-OMe modified ribonucleotides at positions 1, 2, 3, 5, 7, 9,11, 13, 14, 16, 17 and 19 and may further comprise at least one abasicor inverted abasic pseudo-nucleotide for example in position 15. Inother embodiments, (N)x comprises 2′-OMe modified ribonucleotides atpositions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and may further comprise atleast one abasic or inverted abasic pseudo-nucleotide for example inposition 7. In other embodiments, (N)x comprises 2′-OMe sugar modifiedribonucleotides at positions 2, 4, 6, 11, 13, 15, 17 and 19 and mayfurther comprise at least one abasic or inverted abasicpseudo-nucleotide for example in position 8. In other embodiments, (N)xcomprises 2′-OMe sugar modified ribonucleotides at positions 2, 4, 6, 8,11, 13, 15, 17 and 19 and may further comprise at least one abasic orinverted abasic pseudo-nucleotide for example in position 9. In otherembodiments, (N)x comprises 2′-OMe sugar modified ribonucleotides atpositions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and may further comprise atleast one abasic or inverted abasic pseudo-nucleotide for example inposition 10. In other embodiments, (N)x comprises 2′-OMe sugar modifiedribonucleotides at positions 2, 4, 6, 8, 13, 15, 17 and 19 and mayfurther comprise at least one abasic or inverted abasicpseudo-nucleotide for example in position 11. In other embodiments, (N)xcomprises 2′-OMe sugar modified ribonucleotides at positions 2, 4, 6, 8,11, 13, 15, 17 and 19 and may further comprise at least one abasic orinverted abasic pseudo-nucleotide for example in position 12. In otherembodiments, (N)x comprises 2′-OMe sugar modified ribonucleotides atpositions 2, 4, 6, 8, 11, 15, 17 and 19 and may further comprise atleast one abasic or inverted abasic pseudo-nucleotide for example inposition 13.

In yet other embodiments (N)x comprises at least one nucleotide mismatchrelative to the target gene. In certain preferred embodiments, (N)xcomprises a single nucleotide mismatch on position 5, 6, or 14. In oneembodiment of Structure (C), at least two nucleotides at either or boththe 5′ and 3′ termini of (N′)y are joined by a 2′-5′ phosphodiesterbond. In certain preferred embodiments x=y=19 or x=y=23; in (N)x thenucleotides alternate between 2′-OMe sugar modified ribonucleotides andunmodified ribonucleotides, and the ribonucleotide located at the middleof (N)x being unmodified; and three nucleotides at the 3′ terminus of(N′)y are joined by two 2′-5′ phosphodiester bonds (set forth herein asStructure I). In other preferred embodiments, x=y=19 or x=y=23; in (N)xthe nucleotides alternate between 2′-OMe sugar modified ribonucleotidesand unmodified ribonucleotides, and the ribonucleotide located at themiddle of (N)x being unmodified; and four consecutive nucleotides at the5′ terminus of (N′)_(y) are joined by three 2′-5′ phosphodiester bonds.In a further embodiment, an additional nucleotide located in the middleposition of (N)y is 2′-OMe sugar modified. In another preferredembodiment, in (N)x the nucleotides alternate between 2′-OMe sugarmodified ribonucleotides and unmodified ribonucleotides, and in (N′)yfour consecutive nucleotides at the 5′ terminus are joined by three2′-5′ phosphodiester bonds and the 5′ terminal nucleotide or two orthree consecutive nucleotides at the 5′ terminus comprise 3′-O-methyl(3′-OMe) modifications.

In certain preferred embodiments of Structure C, x=y=19 and in (N′)y, atleast one position comprises an abasic or inverted abasicpseudo-nucleotide, preferably five positions comprises an abasic orinverted abasic pseudo-nucleotides. In various embodiments, thefollowing positions comprise an abasic or inverted abasic: positions 1and 16-19, positions 15-19, positions 1-2 and 17-19, positions 1-3 and18-19, positions 1-4 and 19 and positions 1-5. (N′)y may furthercomprise at least one LNA nucleotide.

In certain preferred embodiments of Structure C, x=y=19 and in (N′)y thenucleotide in at least one position comprises a mirror nucleotide, adeoxyribonucleotide and a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond.

In certain preferred embodiments of Structure C, x=y=19 and (N′)ycomprises a mirror nucleotide. In various embodiments the mirrornucleotide is an L-DNA nucleotide. In certain embodiments the L-DNA isL-deoxyribocytidine. In some embodiments (N′)y comprises L-DNA atposition 18. In other embodiments (N′)y comprises L-DNA at positions 17and 18. In certain embodiments (N′)y comprises L-DNA substitutions atpositions 2 and at one or both of positions 17 and 18. In certainembodiments (N′)y further comprises a 5′ terminal cap nucleotide such as5′-O-methyl DNA or an abasic or inverted abasic pseudo-nucleotide as anoverhang.

In yet other embodiments (N′)y comprises at least one nucleotidemismatch relative to the target gene. In certain preferred embodiments,(N′)y comprises a single nucleotide mismatch on position 6, 14, or 15.

In yet other embodiments (N′)y comprises a DNA at position 15 and L-DNAat one or both of positions 17 and 18. In that structure, position 2 mayfurther comprise an L-DNA or an abasic pseudo-nucleotide.

Other embodiments of Structure C are envisaged wherein x=y=21 or whereinx=y=23; in these embodiments the modifications for (N′)y discussed aboveinstead of being on positions 15, 16, 17, 18 are on positions 17, 18,19, 20 for 21 mer and on positions 19, 20, 21, 22 for 23 mer; similarlythe modifications at one or both of positions 17 and 18 are on one orboth of positions 19 or 20 for a 21 mer and one or both of positions 21and 22 for a 23 mer. All modifications in the 19 mer are similarlyadjusted for the 21 and 23 mers.

According to various embodiments of Structure (C), in (N′)y 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides at the 3′terminus are linked by 2′-5′ internucleotide linkages In one preferredembodiment, four consecutive nucleotides at the 3′ terminus of (N′)y arejoined by three 2′-5′ phosphodiester bonds, wherein one or more of the2′-5′ nucleotides which form the 2′-5′ phosphodiester bonds furthercomprises a 3′-O-methyl sugar modification. Preferably the 3′ terminalnucleotide of (N′)y comprises a 2′-O-methyl sugar modification. Incertain preferred embodiments of Structure C, x=y=19 and in (N′)y two ormore consecutive nucleotides at positions 15, 16, 17, 18 and 19 comprisea nucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidebond. In various embodiments the nucleotide forming the 2′-5′internucleotide bond comprises a 3′ deoxyribose nucleotide or a 3′methoxy nucleotide. In some embodiments the nucleotides at positions 17and 18 in (N′)y are joined by a 2′-5′ internucleotide bond. In otherembodiments the nucleotides at positions 16, 17, 18, 16-17, 17-18, or16-18 in (N′)y are joined by a 2′-5′ internucleotide bond.

In certain embodiments (N′)y comprises an L-DNA at position 2 and 2′-5′internucleotide bonds at positions 16, 17, 18, 16-17, 17-18, or 16-18.In certain embodiments (N′)y comprises 2′-5′ internucleotide bonds atpositions 16, 17, 18, 16-17, 17-18, or 16-18 and a 5′ terminal capnucleotide.

According to various embodiments of Structure (C), in (N′) y 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides at eitherterminus or 2-8 modified nucleotides at each of the 5′ and 3′ terminiare independently mirror nucleotides. In some embodiments the mirrornucleotide is an L-ribonucleotide. In other embodiments the mirrornucleotide is an L-deoxyribonucleotide. The mirror nucleotide mayfurther be modified at the sugar or base moiety or in an internucleotidelinkage.

In one preferred embodiment of Structure (C), the 3′ terminal nucleotideor two or three consecutive nucleotides at the 3′ terminus of (N′)y areL-deoxyribonucleotides.

In other embodiments of Structure (C), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides at either terminus or2-8 modified nucleotides at each of the 5′ and 3′ termini areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe).

In one series of preferred embodiments, three, four or five consecutivenucleotides at the 5′ terminus of (N′)y comprise the 2′-OMemodification. In another preferred embodiment, three consecutivenucleotides at the 3′ terminus of (N′)y comprise the 2′-O-methylmodification.

In some embodiments of Structure (C), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides at either or 2-8modified nucleotides at each of the 5′ and 3′ termini are independentlybicyclic nucleotide. In various embodiments the bicyclic nucleotide is alocked nucleic acid (LNA). A 2′-O,4′-C-ethylene-bridged nucleic acid(ENA) is a species of LNA (see below).

In various embodiments (N′)y comprises modified nucleotides at the 5′terminus or at both the 3′ and 5′ termini.

In some embodiments of Structure (C), at least two nucleotides at eitheror both the 5′ and 3′ termini of (N′)y are joined by P-ethoxy backbonemodifications. In certain preferred embodiments x=y=19 or x=y=23; in(N)x the nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle position of (N)x being unmodified; and four consecutivenucleotides at the 3′ terminus or at the 5′ terminus of (N′)y are joinedby three P-ethoxy backbone modifications. In another preferredembodiment, three consecutive nucleotides at the 3′ terminus or at the5′ terminus of (N′)y are joined by two P-ethoxy backbone modifications.

In some embodiments of Structure (C), in (N′)y 2, 3, 4, 5, 6, 7 or 8,consecutive ribonucleotides at each of the 5′ and 3′ termini areindependently mirror nucleotides, nucleotides joined by 2′-5′phosphodiester bond, 2′ sugar modified nucleotides or bicyclicnucleotide. In one embodiment, the modification at the 5′ and 3′ terminiof (N′)y is identical. In one preferred embodiment, four consecutivenucleotides at the 5′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds and three consecutive nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. Inanother embodiment, the modification at the 5′ terminus of (N′)y isdifferent from the modification at the 3′ terminus of (N′)y. In onespecific embodiment, the modified nucleotides at the 5′ terminus of(N′)y are mirror nucleotides and the modified nucleotides at the 3′terminus of (N′)y are joined by 2′-5′ phosphodiester bond. In anotherspecific embodiment, three consecutive nucleotides at the 5′ terminus of(N′)y are LNA nucleotides and three consecutive nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. In (N)xthe nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle of (N)x being unmodified, or the ribonucleotides in (N)xbeing unmodified

In another embodiment of Structure (C), the present invention provides acompound wherein x=y=19 or x=y=23; in (N)x the nucleotides alternatebetween modified ribonucleotides and unmodified ribonucleotides, eachmodified ribonucleotide being modified so as to have a 2′-O-methyl onits sugar and the ribonucleotide located at the middle of (N)x beingunmodified; three nucleotides at the 3′ terminus of (N′)y are joined bytwo 2′-5′ phosphodiester bonds and three nucleotides at the 5′ terminusof (N′)y are LNA such as ENA.

In another embodiment of Structure (C), five consecutive nucleotides atthe 5′ terminus of (N′)y comprise the 2′-O-methyl sugar modification andtwo consecutive nucleotides at the 3′ terminus of (N′)y are L-DNA.

In yet another embodiment, the present invention provides a compoundwherein x=y=19 or x=y=23; (N)x consists of unmodified ribonucleotides;three consecutive nucleotides at the 3′ terminus of (N′)y are joined bytwo 2′-5′ phosphodiester bonds and three consecutive nucleotides at the5′ terminus of (N′)y are LNA such as ENA.

According to other embodiments of Structure (C), in (N′)y the 5′ or 3′terminal nucleotide, or 2, 3, 4, 5 or 6 consecutive nucleotides ateither termini or 1-4 modified nucleotides at each of the 5′ and 3′termini are independently phosphonocarboxylate or phosphinocarboxylatenucleotides (PACE nucleotides). In some embodiments the PACE nucleotidesare deoxyribonucleotides. In some preferred embodiments in (N′)y, 1 or 2consecutive nucleotides at each of the 5′ and 3′ termini are PACEnucleotides. Examples of PACE nucleotides and analogs are disclosed inU.S. Pat. Nos. 6,693,187 and 7,067,641 both incorporated by reference.

In additional embodiments, the present invention provides a compoundhaving Structure (D):

(D) 5′ (N)x-Z 3′ antisense strand 3′ Z′-(N′)y 5′ sense strandwherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide or a modified deoxyribonucleotide;wherein each of (N)x and (N′)y is an oligomer in which each consecutivenucleotide is joined to the next nucleotide by a covalent bond and eachof x and y is an integer between 18 and 40;wherein (N)x comprises unmodified ribonucleotides further comprising onemodified nucleotide at the 3′ terminal or penultimate position, whereinthe modified nucleotide is selected from the group consisting of abicyclic nucleotide, a 2′ sugar modified nucleotide, a mirrornucleotide, an altritol nucleotide, or a nucleotide joined to anadjacent nucleotide by an internucleotide linkage selected from a 2′-5′phosphodiester bond, a P-alkoxy linkage or a PACE linkage;wherein (N′)y comprises unmodified ribonucleotides further comprisingone modified nucleotide at the 5′ terminal or penultimate position,wherein the modified nucleotide is selected from the group consisting ofa bicyclic nucleotide, a 2′ sugar modified nucleotide, a mirrornucleotide, an altritol nucleotide, or a nucleotide joined to anadjacent nucleotide by an internucleotide linkage selected from a 2′-5′phosphodiester bond, a P-alkoxy linkage or a PACE linkage;wherein in each of (N)x and (N′)y modified and unmodified nucleotidesare not alternating;wherein each of Z and Z′ may be present or absent, but if present is 1-5deoxyribonucleotides covalently attached at the 3′ terminus of anyoligomer to which it is attached;wherein the sequence of (N′)y is a sequence substantially complementaryto (N)x; and wherein the sequence of (N)_(x) comprises an antisensesequence having substantial complementarity to about 18 to about 40consecutive ribonucleotides in the mRNA set forth in any one of SEQ IDNOS:1-58.

In one embodiment of Structure (D), x=y=19 or x=y=23; (N)x comprisesunmodified ribonucleotides in which two consecutive nucleotides linkedby one 2′-5′ internucleotide linkage at the 3′ terminus; and (N′)ycomprises unmodified ribonucleotides in which two consecutivenucleotides linked by one 2′-5′ internucleotide linkage at the 5′terminus.

In some embodiments, x=y=19 or x=y=23; (N)x comprises unmodifiedribonucleotides in which three consecutive nucleotides at the 3′terminus are joined together by two 2′-5′ phosphodiester bonds; and(N′)y comprises unmodified ribonucleotides in which four consecutivenucleotides at the 5′ terminus are joined together by three 2′-5′phosphodiester bonds (set forth herein as Structure II).

According to various embodiments of Structure (D) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 3′ terminus of (N)x and 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides startingat the ultimate or penultimate position of the 5′ terminus of (N′)y arelinked by 2′-5′ internucleotide linkages.

According to one preferred embodiment of Structure (D), four consecutivenucleotides at the 5′ terminus of (N′)y are joined by three 2′-5′phosphodiester bonds and three consecutive nucleotides at the 3′terminus of (N′)x are joined by two 2′-5′ phosphodiester bonds. Threenucleotides at the 5′ terminus of (N′)y and two nucleotides at the 3′terminus of (N′)x may also comprise 3′-O-methyl modifications.

According to various embodiments of Structure (D), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides starting at the ultimateor penultimate position of the 3′ terminus of (N)x and 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N′)y areindependently mirror nucleotides. In some embodiments the mirror is anL-ribonucleotide. In other embodiments the mirror nucleotide isL-deoxyribonucleotide.

In other embodiments of Structure (D), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N)x and 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N′)y areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe).

In one preferred embodiment of Structure (D), five consecutivenucleotides at the 5′ terminus of (N′)y comprise the 2′-O-methylmodification and five consecutive nucleotides at the 3′ terminus of(N′)x comprise the 2′-O-methyl modification. In another preferredembodiment of Structure (D), ten consecutive nucleotides at the 5′terminus of (N′)y comprise the 2′-O-methyl modification and fiveconsecutive nucleotides at the 3′ terminus of (N′)x comprise the2′-β-methyl modification. In another preferred embodiment of Structure(D), thirteen consecutive nucleotides at the 5′ terminus of (N′)ycomprise the 2′-O-methyl modification and five consecutive nucleotidesat the 3′ terminus of (N′)x comprise the 2′-O-methyl modification.

In some embodiments of Structure (D), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 3′ terminus of (N)x and 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive ribonucleotides startingat the ultimate or penultimate position of the 5′ terminus of (N′)y areindependently a bicyclic nucleotide. In various embodiments the bicyclicnucleotide is a locked nucleic acid (LNA) such as a2′-O,4′-C-ethylene-bridged nucleic acid (ENA).

In various embodiments of Structure (D), (N′)y comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkage;

In various embodiments of Structure (D), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkage;

In embodiments wherein each of the 3′ and 5′ termini of the same strandcomprises a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In one specific embodiment of Structure (D), five consecutivenucleotides at the 5′ terminus of (N′)y comprise the 2′-O-methylmodification and two consecutive nucleotides at the 3′ terminus of (N′)yare L-DNA. In addition, the compound may further comprise fiveconsecutive 2′-β-methyl modified nucleotides at the 3′ terminus of(N′)x.

In various embodiments of Structure (D), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (E):

(E) 5′ (N)x-Z 3′ antisense strand 3′ Z′-(N′)y 5′ sense strandwherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide or a modified deoxyribonucleotide;wherein each of (N)x and (N′)y is an oligomer in which each consecutivenucleotide is joined to the next nucleotide by a covalent bond and eachof x and y is an integer between 18 and 40;wherein (N)x comprises unmodified ribonucleotides further comprising onemodified nucleotide at the 5′ terminal or penultimate position, whereinthe modified nucleotide is selected from the group consisting of abicyclic nucleotide, a 2′ sugar modified nucleotide, a mirrornucleotide, an altritol nucleotide, or a nucleotide joined to anadjacent nucleotide by an internucleotide linkage selected from a 2′-5′phosphodiester bond, a P-alkoxy linkage or a PACE linkage;wherein (N′)y comprises unmodified ribonucleotides further comprisingone modified nucleotide at the 3′ terminal or penultimate position,wherein the modified nucleotide is selected from the group consisting ofa bicyclic nucleotide, a 2′ sugar modified nucleotide, a mirrornucleotide, an altritol nucleotide, or a nucleotide joined to anadjacent nucleotide by an internucleotide linkage selected from a 2′-5′phosphodiester bond, a P-alkoxy linkage or a PACE linkage;wherein in each of (N)x and (N′)y modified and unmodified nucleotidesare not alternating;wherein each of Z and Z′ may be present or absent, but if present is 1-5deoxyribonucleotides covalently attached at the 3′ terminus of anyoligomer to which it is attached;wherein the sequence of (N′)y is a sequence substantially complementaryto (N)x; and wherein the sequence of (N)_(x) comprises an antisensesequence having substantial complementarity to about 18 to about 40consecutive ribonucleotides in the mRNA set forth in any one of SEQ IDNOS:1-58.

In certain preferred embodiments the ultimate nucleotide at the 5′terminus of (N)x is unmodified.

According to various embodiments of Structure (E) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N)x, preferablystarting at the 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N′)y are linked by 2′-5′internucleotide linkages.

According to various embodiments of Structure (E), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides starting at the ultimateor penultimate position of the 5′ terminus of (N)x, preferably startingat the 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 consecutive nucleotides starting at the ultimate or penultimateposition of the 3′ terminus of (N′)y are independently mirrornucleotides. In some embodiments the mirror is an L-ribonucleotide. Inother embodiments the mirror nucleotide is L-deoxyribonucleotide.

In other embodiments of Structure (E), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 5′ terminus of (N)x, preferably starting atthe 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N′)y are independently 2′sugar modified nucleotides. In some embodiments the 2′ sugarmodification comprises the presence of an amino, a fluoro, an alkoxy oran alkyl moiety. In certain embodiments the 2′ sugar modificationcomprises a methoxy moiety (2′-OMe).

In some embodiments of Structure (E), in (N′)y 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive ribonucleotides starting at theultimate or penultimate position of the 5′ terminus of (N)x, preferablystarting at the 5′ penultimate position, and 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 consecutive ribonucleotides starting at the ultimate orpenultimate position of the 3′ terminus of (N′)y are independently abicyclic nucleotide. In various embodiments the bicyclic nucleotide is alocked nucleic acid (LNA) such as a 2′-O,4′-C-ethylene-bridged nucleicacid (ENA).

In various embodiments of Structure (E), (N′)y comprises modifiednucleotides selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, a nucleotidejoined to an adjacent nucleotide by a P-alkoxy backbone modification ora nucleotide joined to an adjacent nucleotide by an internucleotidelinkage selected from a 2′-5′ phosphodiester bond, a P-alkoxy linkage ora PACE linkage at the 3′ terminus or at each of the 3′ and 5′ termini.

In various embodiments of Structure (E), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 5′ terminus or at each of the 3′ and 5′ termini.

In one embodiment where both 3′ and 5′ termini of the same strandcomprise a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In various embodiments of Structure (E), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (F):

(F) 5′ (N)x-Z 3′ antisense strand 3′ Z′-(N′)y 5′ sense strandwherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide or a modified deoxyribonucleotide;wherein each of (N)x and (N′)y is an oligomer in which each consecutivenucleotide is joined to the next nucleotide by a covalent bond and eachof x and y is an integer between 18 and 40;wherein each of (N)x and (N′)y comprise unmodified ribonucleotides inwhich each of (N)x and (N′)y independently comprise one modifiednucleotide at the 3′ terminal or penultimate position wherein themodified nucleotide is selected from the group consisting of a bicyclicnucleotide, a 2′ sugar modified nucleotide, a mirror nucleotide, anucleotide joined to an adjacent nucleotide by a P-alkoxy backbonemodification or a nucleotide joined to an adjacent nucleotide by a 2′-5′phosphodiester bond;wherein in each of (N)x and (N′)y modified and unmodified nucleotidesare not alternating;wherein each of Z and Z′ may be present or absent, but if present is 1-5deoxyribonucleotides covalently attached at the 3′ terminus of anyoligomer to which it is attached;wherein the sequence of (N′)y is a sequence substantially complementaryto (N)x; and wherein the sequence of (N)_(x) comprises an antisensesequence having substantial complementarity to about 18 to about 40consecutive ribonucleotides in mRNA set forth in any one of SEQ IDNOS:1-58.

In some embodiments of Structure (F), x=y=19 or x=y=23; (N′)y comprisesunmodified ribonucleotides in which two consecutive nucleotides at the3′ terminus comprises two consecutive mirror deoxyribonucleotides; and(N)x comprises unmodified ribonucleotides in which one nucleotide at the3′ terminus comprises a mirror deoxyribonucleotide (set forth asStructure III).

According to various embodiments of Structure (F) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independentlybeginning at the ultimate or penultimate position of the 3′ termini of(N)x and (N′)y are linked by 2′-5′ internucleotide linkages.

According to one preferred embodiment of Structure (F), threeconsecutive nucleotides at the 3′ terminus of (N′)y are joined by two2′-5′ phosphodiester bonds and three consecutive nucleotides at the 3′terminus of (N′)x are joined by two 2′-5′ phosphodiester bonds.

According to various embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides independently beginningat the ultimate or penultimate position of the 3′ termini of (N)x and(N′)y are independently mirror nucleotides. In some embodiments themirror nucleotide is an L-ribonucleotide. In other embodiments themirror nucleotide is an L-deoxyribonucleotide.

In other embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ termini of (N)x and (N′)y areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe).

In some embodiments of Structure (F), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ termini of (N)x and (N′)y areindependently a bicyclic nucleotide. In various embodiments the bicyclicnucleotide is a locked nucleic acid (LNA) such as a2′-O,4′-C-ethylene-bridged nucleic acid (ENA).

In various embodiments of Structure (F), (N′)y comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 3′ terminus or at both the 3′ and 5′ termini.

In various embodiments of Structure (F), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 3′ terminus or at each of the 3′ and 5′ termini.

In one embodiment where each of 3′ and 5′ termini of the same strandcomprise a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In various embodiments of Structure (F), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure

(G) 5′ (N)x-Z 3′ antisense strand 3′ Z′-(N′)y 5′ sense strandwherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide or a modified deoxyribonucleotide;wherein each of (N)x and (N′)y is an oligomer in which each consecutivenucleotide is joined to the next nucleotide by a covalent bond and eachof x and y is an integer between 18 and 40;wherein each of (N)x and (N′)y comprise unmodified ribonucleotides inwhich each of (N)x and (N′)y independently comprise one modifiednucleotide at the 5′ terminal or penultimate position wherein themodified nucleotide is selected from the group consisting of a bicyclicnucleotide, a 2′ sugar modified nucleotide, a mirror nucleotide, anucleotide joined to an adjacent nucleotide by a P-alkoxy backbonemodification or a nucleotide joined to an adjacent nucleotide by a 2′-5′phosphodiester bond;wherein for (N)x the modified nucleotide is preferably at penultimateposition of the 5′ terminal;wherein in each of (N)x and (N′)y modified and unmodified nucleotidesare not alternating;wherein each of Z and Z′ may be present or absent, but if present is 1-5deoxyribonucleotides covalently attached at the 3′ terminus of anyoligomer to which it is attached;wherein the sequence of (N′)_(y) is a sequence substantiallycomplementary to (N)x; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in the mRNA set forth in any one ofSEQ ID NOS:1-58.

In some embodiments of Structure (G), x=y=19.

According to various embodiments of Structure (G) 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive ribonucleotides independentlybeginning at the ultimate or penultimate position of the 5′ termini of(N)x and (N′)y are linked by 2′-5′ internucleotide linkages. For (N)xthe modified nucleotides preferably starting at the penultimate positionof the 5′ terminal.

According to various embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 or 14 consecutive nucleotides independently beginningat the ultimate or penultimate position of the 5′ termini of (N)x and(N′)y are independently mirror nucleotides. In some embodiments themirror nucleotide is an L-ribonucleotide. In other embodiments themirror nucleotide is an L-deoxyribonucleotide. For (N)x the modifiednucleotides preferably starting at the penultimate position of the 5′terminal.

In other embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 5′ termini of (N)x and (N′)y areindependently 2′ sugar modified nucleotides. In some embodiments the 2′sugar modification comprises the presence of an amino, a fluoro, analkoxy or an alkyl moiety. In certain embodiments the 2′ sugarmodification comprises a methoxy moiety (2′-OMe). In some preferredembodiments the consecutive modified nucleotides preferably begin at thepenultimate position of the 5′ terminus of (N)x.

In one preferred embodiment of Structure (G), five consecutiveribonucleotides at the 5′ terminus of (N′)y comprise a 2′-O-methylmodification and one ribonucleotide at the 5′ penultimate position of(N′)x comprises a 2′-O-methyl modification. In another preferredembodiment of Structure (G), five consecutive ribonucleotides at the 5′terminus of (N′)y comprise a 2′-O-methyl modification and twoconsecutive ribonucleotides at the 5′ terminal position of (N′)xcomprise a 2′-O-methyl modification.

In some embodiments of Structure (G), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 5′ termini of (N)x and (N′)y arebicyclic nucleotides. In various embodiments the bicyclic nucleotide isa locked nucleic acid (LNA) such as a 2′-O,4′-C-ethylene-bridged nucleicacid (ENA). In some preferred embodiments the consecutive modifiednucleotides preferably begin at the penultimate position of the 5′terminus of (N)x.

In various embodiments of Structure (G), (N′)y comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 5′ terminus or at each of the 3′ and 5′ termini.

In various embodiments of Structure (G), (N)x comprises a modifiednucleotide selected from a bicyclic nucleotide, a 2′ sugar modifiednucleotide, a mirror nucleotide, an altritol nucleotide, or a nucleotidejoined to an adjacent nucleotide by an internucleotide linkage selectedfrom a 2′-5′ phosphodiester bond, a P-alkoxy linkage or a PACE linkageat the 5′ terminus or at each of the 3′ and 5′ termini.

In one embodiment where each of 3′ and 5′ termini of the same strandcomprise a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.In various embodiments of Structure (G), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In additional embodiments, the present invention provides a compoundhaving Structure (H):

(H) 5′ (N)x-Z 3′ antisense strand 3′ Z′-(N′)y 5′ sense strandwherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide or a modified deoxyribonucleotide;wherein each of (N)x and (N′)y is an oligomer in which each consecutivenucleotide is joined to the next nucleotide by a covalent bond and eachof x and y is an integer between 18 and 40;wherein (N)x comprises unmodified ribonucleotides further comprising onemodified nucleotide at the 3′ terminal or penultimate position or the 5′terminal or penultimate position, wherein the modified nucleotide isselected from the group consisting of a bicyclic nucleotide, a 2′ sugarmodified nucleotide, a mirror nucleotide, an altritol nucleotide, or anucleotide joined to an adjacent nucleotide by an internucleotidelinkage selected from a 2′-5′ phosphodiester bond, a P-alkoxy linkage ora PACE linkage;wherein (N′)y comprises unmodified ribonucleotides further comprisingone modified nucleotide at an internal position, wherein the modifiednucleotide is selected from the group consisting of a bicyclicnucleotide, a 2′ sugar modified nucleotide, a mirror nucleotide, analtritol nucleotide, or a nucleotide joined to an adjacent nucleotide byan internucleotide linkage selected from a 2′-5′ phosphodiester bond, aP-alkoxy linkage or a PACE linkage;wherein in each of (N)x and (N′)y modified and unmodified nucleotidesare not alternating;wherein each of Z and Z′ may be present or absent, but if present is 1-5deoxyribonucleotides covalently attached at the 3′ terminus of anyoligomer to which it is attached;wherein the sequence of (N′)_(y) is a sequence substantiallycomplementary to (N)x; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58.

In one embodiment of Structure (H), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ terminus or the 5′ terminusor both termini of (N)x are independently 2′ sugar modified nucleotides,bicyclic nucleotides, mirror nucleotides, altritol nucleotides ornucleotides joined to an adjacent nucleotide by a 2′-5′ phosphodiesterbond and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutiveinternal ribonucleotides in (N′)y are independently 2′ sugar modifiednucleotides, bicyclic nucleotides, mirror nucleotides, altritolnucleotides or nucleotides joined to an adjacent nucleotide by a 2′-5′phosphodiester bond. In some embodiments the 2′ sugar modificationcomprises the presence of an amino, a fluoro, an alkoxy or an alkylmoiety. In certain embodiments the 2′ sugar modified ribonucleotidecomprises a methoxy moiety (2′-OMe).

In another embodiment of Structure (H), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 consecutive ribonucleotides independently beginning at theultimate or penultimate position of the 3′ terminus or the 5′ terminusor 2-8 consecutive nucleotides at each of 5′ and 3′ termini of (N′)y areindependently 2′ sugar modified nucleotides, bicyclic nucleotides,mirror nucleotides, altritol nucleotides or nucleotides joined to anadjacent nucleotide by a 2′-5′ phosphodiester bond, and 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 consecutive internal ribonucleotides in(N)x are independently 2′ sugar modified nucleotides, bicyclicnucleotides, mirror nucleotides, altritol nucleotides or nucleotidesjoined to an adjacent nucleotide by a 2′-5′ phosphodiester bond.

In one embodiment wherein each of 3′ and 5′ termini of the same strandcomprises a modified nucleotide, the modification at the 5′ and 3′termini is identical. In another embodiment, the modification at the 5′terminus is different from the modification at the 3′ terminus of thesame strand. In one specific embodiment, the modified nucleotides at the5′ terminus are mirror nucleotides and the modified nucleotides at the3′ terminus of the same strand are joined by 2′-5′ phosphodiester bond.

In various embodiments of Structure (H), the modified nucleotides in(N)x are different from the modified nucleotides in (N′)y. For example,the modified nucleotides in (N)x are 2′ sugar modified nucleotides andthe modified nucleotides in (N′)y are nucleotides linked by 2′-5′internucleotide linkages. In another example, the modified nucleotidesin (N)x are mirror nucleotides and the modified nucleotides in (N′)y arenucleotides linked by 2′-5′ internucleotide linkages. In anotherexample, the modified nucleotides in (N)x are nucleotides linked by2′-5′ internucleotide linkages and the modified nucleotides in (N′)y aremirror nucleotides.

In one preferred embodiment of Structure (H), x=y=19; three consecutiveribonucleotides at the 9-11 nucleotide positions 9-11 of (N′)y comprise2′-O-methyl modification and five consecutive ribonucleotides at the 3′terminal position of (N′)x comprise 2′-O-methyl modification.

In one aspect the present invention provides a compound having Structure(I) set forth below:

(I) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ may be present or absent, but if present isindependently 1-5 consecutive nucleotides covalently attached at the 3′terminus of the strand in which it is present;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein x=18 to 27;wherein y=18 to 27;wherein (N)x comprises modified and unmodified ribonucleotides, eachmodified ribonucleotide having a 2′-O-methyl on its sugar, wherein N atthe 3′ terminus of (N)x is a modified ribonucleotide, (N)x comprises atleast five alternating modified ribonucleotides beginning at the 3′ endand at least nine modified ribonucleotides in total and each remaining Nis an unmodified ribonucleotide;wherein in (N′)y at least one unconventional moiety is present, whichunconventional moiety may be an abasic ribose moiety, an abasicdeoxyribose moiety, a modified or unmodified deoxyribonucleotide, amirror nucleotide, and a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide phosphate bond; andwherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58.

In some embodiments x=y=19. In other embodiments x=y=23. In someembodiments the at least one unconventional moiety is present atpositions 15, 16, 17, or 18 in (N′)y. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some preferredembodiments the unconventional moiety is a mirror nucleotide, preferablyan L-DNA moiety. In some embodiments an L-DNA moiety is present atposition 17, position 18 or positions 17 and 18.

In other embodiments the unconventional moiety is an abasic moiety. Invarious embodiments (N′)y comprises at least five abasic ribose moietiesor abasic deoxyribose moieties.

In yet other embodiments (N′)y comprises at least five abasic ribosemoieties or abasic deoxyribose moieties and at least one of N′ is anLNA.

In some embodiments of Structure (IX) (N)x comprises nine alternatingmodified ribonucleotides. In other embodiments of Structure (I) (N)xcomprises nine alternating modified ribonucleotides further comprising a2′O modified nucleotide at position 2. In some embodiments (N)xcomprises 2′-OMe sugar modified ribonucleotides at the odd numberedpositions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19. In other embodiments (N)xfurther comprises a 2′-OMe sugar modified ribonucleotide at one or bothof positions 2 and 18. In yet other embodiments (N)x comprises 2′-OMesugar modified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17,19.

In various embodiments z″ is present and is selected from an abasicribose moiety, a deoxyribose moiety; an inverted abasic ribose moiety, adeoxyribose moiety; C6-amino-Pi; a mirror nucleotide.

In another aspect the present invention provides a compound havingStructure (J) set forth below:

(J) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ may be present or absent, but if present isindependently 1-5 consecutive nucleotides covalently attached at the 3′terminus of the strand in which it is present;wherein z″ may be present or absent but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein x=18 to 27;wherein y=18 to 27;wherein (N)x comprises modified or unmodified ribonucleotides, andoptionally at least one unconventional moiety;wherein in (N′)y at least one unconventional moiety is present, whichunconventional moiety may be an abasic ribose moiety, an abasicdeoxyribose moiety, a modified or unmodified deoxyribonucleotide, amirror nucleotide, a non-base pairing nucleotide analog or a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide phosphatebond; andwherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58.

In some embodiments x=y=19. In other embodiments x=y=23. In somepreferred embodiments (N)x comprises modified and unmodifiedribonucleotides, and at least one unconventional moiety.

In some embodiments in (N)x the N at the 3′ terminus is a modifiedribonucleotide and (N)x comprises at least 8 modified ribonucleotides.In other embodiments at least 5 of the at least 8 modifiedribonucleotides are alternating beginning at the 3′ end. In someembodiments (N)x comprises an abasic moiety in one of positions 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15.

In some embodiments the at least one unconventional moiety in (N′)y ispresent at positions 15, 16, 17, or 18. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some preferredembodiments the unconventional moiety is a mirror nucleotide, preferablyan L-DNA moiety. In some embodiments an L-DNA moiety is present atposition 17, position 18 or positions 17 and 18. In other embodimentsthe at least one unconventional moiety in (N′)y is an abasic ribosemoiety or an abasic deoxyribose moiety.

In various embodiments of Structure (X) z″ is present and is selectedfrom an abasic ribose moiety, a deoxyribose moiety; an inverted abasicribose moiety, a deoxyribose moiety; C6-amino-Pi; a mirror nucleotide.

In yet another aspect the present invention provides a compound havingStructure (K) set forth below:

(K) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y)-z″ 5′(sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ may be present or absent, but if present isindependently 1-5 consecutive nucleotides covalently attached at the 3′terminus of the strand in which it is present;wherein z″ may be present or absent but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;wherein x=18 to 27;wherein y=18 to 27;wherein (N)x comprises a combination of modified or unmodifiedribonucleotides and unconventional moieties, any modified ribonucleotidehaving a 2′-O-methyl on its sugar;wherein (N′)y comprises modified or unmodified ribonucleotides andoptionally an unconventional moiety, any modified ribonucleotide havinga 2′ OMe on its sugar;wherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58; and wherein there are less than 15 consecutive nucleotidescomplementary to the mRNA.

In some embodiments x=y=19. In other embodiments x=y=23. In somepreferred embodiments the at least one preferred one unconventionalmoiety is present in (N)x and is an abasic ribose moiety or an abasicdeoxyribose moiety. In other embodiments the at least one unconventionalmoiety is present in (N)x and is a non-base pairing nucleotide analog.In various embodiments (N′)y comprises unmodified ribonucleotides. Insome embodiments (N)x comprises at least five abasic ribose moieties orabasic deoxyribose moieties or a combination thereof. In certainembodiments (N)x and/or (N′)y comprise modified ribonucleotides which donot base pair with corresponding modified or unmodified ribonucleotidesin (N′)y and/or (N)x.

In various embodiments the present invention provides an siRNA set forthin Structure (L):

(L) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)wherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide and a modified deoxyribonucleotide;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ are absent;wherein x=y=19;wherein in (N′)y the nucleotide in at least one of positions 15, 16, 17,18 and 19 comprises a nucleotide selected from an abasicpseudo-nucleotide, a mirror nucleotide, a deoxyribonucleotide and anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidebond;wherein (N)x comprises alternating modified ribonucleotides andunmodified ribonucleotides each modified ribonucleotide being modifiedso as to have a 2′-O-methyl on its sugar and the ribonucleotide locatedat the middle position of (N)x being modified or unmodified, preferablyunmodified; andwherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58.

In some embodiments of Structure (L), in (N′)y the nucleotide in one orboth of positions 17 and 18 comprises a modified nucleotide selectedfrom an abasic pseudo-nucleotide, a mirror nucleotide and a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide bond. Insome embodiments the mirror nucleotide is selected from L-DNA and L-RNA.In various embodiments the mirror nucleotide is L-DNA.

In various embodiments (N′)y comprises a modified nucleotide at position15 wherein the modified nucleotide is selected from a mirror nucleotideand a deoxyribonucleotide.

In certain embodiments (N′)y further comprises a modified nucleotide orpseudo nucleotide at position 2 wherein the pseudo nucleotide may be anabasic pseudo-nucleotide analog and the modified nucleotide isoptionally a mirror nucleotide.

In various embodiments the antisense strand (N)x comprises 2′O-Memodified ribonucleotides at the odd numbered positions (5′ to 3′;positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). In some embodiments (N)xfurther comprises 2′O-Me modified ribonucleotides at one or bothpositions 2 and 18. In other embodiments (N)x comprises 2′-OMe sugarmodified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

Other embodiments of Structures (L), (I) and (J) are envisaged whereinx=y=21 or wherein x=y=23; in these embodiments the modifications for(N′)y discussed above instead of being in positions 17 and 18 are inpositions 19 and 20 for 21-mer oligonucleotide and 21 and 22 for 23 meroligonucleotide; similarly the modifications in positions 15, 16, 17, 18or 19 are in positions 17, 18, 19, 20 or 21 for the 21-meroligonucleotide and positions 19, 20, 21, 22, or 23 for the 23-meroligonucleotide. The 2′-OMe modifications on the antisense strand aresimilarly adjusted. In some embodiments (N)x comprises 2′-OMe sugarmodified ribonucleotides at the odd numbered positions (5′ to 3′;positions 1, 3, 5, 7, 9, 12, 14, 16, 18, 20 for the 21 meroligonucleotide [nucleotide at position 11 unmodified] and 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23 for the 23 mer oligonucleotide [nucleotideat position 12 unmodified]. In other embodiments (N)x comprises 2′-OMesugar modified ribonucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16,18, 20 [nucleotide at position 11 unmodified for the 21 meroligonucleotide and at positions 2, 4, 6, 8, 10, 13, 15, 17, 19, 21, 23for the 23 mer oligonucleotide [nucleotide at position 12 unmodified].

In some embodiments (N′)y further comprises a 5′ terminal capnucleotide. In various embodiments the terminal cap moiety is selectedfrom an abasic pseudo-nucleotide analog, an inverted abasicpseudo-nucleotide analog, an L-DNA nucleotide, and a C6-imine phosphate(C6 amino linker with phosphate at terminus).

In other embodiments the present invention provides a compound havingStructure (M) set forth below:

5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)wherein each of N and N′ is selected from a pseudo-nucleotide and anucleotide;wherein each nucleotide is selected from an unmodified ribonucleotide, amodified ribonucleotide, an unmodified deoxyribonucleotide and amodified deoxyribonucleotide;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ are absent;wherein x=18 to 27;wherein y=18 to 27;wherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58;wherein at least one of N is selected from an abasic pseudo nucleotide,a non-pairing nucleotide analog and a nucleotide mismatch to the mRNA ofa target gene in a position of (N)x such that (N)x comprises less than15 consecutive nucleotides complementary to the mRNA of a target gene.

In other embodiments the present invention provides a double strandedcompound having Structure (N) set forth below:

(N) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)wherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide and a modified deoxyribonucleotide;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ are absent;wherein each of x and y is an integer between 18 and 40;wherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58;wherein (N)x, (N′)y or (N)x and (N′)y comprise non base-pairing modifiednucleotides such that (N)x and (N′)y form less than 15 base pairs in thedouble stranded compound.

In other embodiments the present invention provides a compound havingStructure (O) set forth below:

(O) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)wherein each of N is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide and a modified deoxyribonucleotide;wherein each of N′ is a nucleotide analog selected from a six memberedsugar nucleotide, seven membered sugar nucleotide, morpholino moiety,peptide nucleic acid and combinations thereof;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ are absent;wherein each of x and y is an integer between 18 and 40;wherein the sequence of (N)x is substantially complementary to thesequence of (N′)_(y); and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58.

In other embodiments the present invention provides a compound havingStructure (P) set forth below:

(P) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)wherein each of N and N′ is a nucleotide selected from an unmodifiedribonucleotide, a modified ribonucleotide, an unmodifieddeoxyribonucleotide and a modified deoxyribonucleotide;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein Z and Z′ are absent;wherein each of x and y is an integer between 18 and 40;wherein one of N or N′ in an internal position of (N)x or (N′)y or oneor more of N or N′ at a terminal position of (N)x or (N′)y comprises anabasic moiety or a 2′ modified nucleotide;wherein the sequence of (N)x is substantially complementary to thesequence of (N′)y; and wherein the sequence of (N)_(x) comprises anantisense sequence having substantial complementarity to about 18 toabout 40 consecutive ribonucleotides in mRNA set forth in any one of SEQID NOS:1-58.

In various embodiments (N′)y comprises a modified nucleotide at position15 wherein the modified nucleotide is selected from a mirror nucleotideand a deoxyribonucleotide.

In certain embodiments (N′)y further comprises a modified nucleotide atposition 2 wherein the modified nucleotide is selected from a mirrornucleotide and an abasic pseudo-nucleotide analog.

In various embodiments the antisense strand (N)x comprises 2′O-Memodified ribonucleotides at the odd numbered positions (5′ to 3′;positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19). In some embodiments (N)xfurther comprises 2′O-Me modified ribonucleotides at one or bothpositions 2 and 18. In other embodiments (N)x comprises 2′-OMe sugarmodified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

The Structural motifs (A)-(P) described above are useful with anyoligonucleotide pair (sense and antisense strands) to a mammalian ornon-mammalian gene. In some embodiments the mammalian gene is a humangene preferably selected from the genes provided in Tables A1-A4, withmRNA set forth in SEQ ID NOS:1-58. In certain preferred embodiments thesense and antisense oligonucleotides of the siRNA are selected from anyone of siRNA pairs set forth in SEQ ID NOS:59-33,596. Table A5 belowshows certain preferred sense and antisense oligonucleotide pairs.

TABLE A5 TARGET SENSE ANTISENSE GENE (N′)y 5′-3′ (N)x 5′-3′ P53GAGAAUAUUUCACCCUUCA UGAAGGGUGAAAUAUUCUC CASP2 GCCAGAAUGUGGAACUCCUAGGAGUUCCACAUUCUGGC RTP801 GUGCCAACCUGAUGCAGCU AGCAGCAUCAGGUUGGCACRTP801 UACUGUAGCAUGAAACAAA UUUGUUUCAUGCUACAGUA RTP801CAGUACUGUAGCAUGAAAC GUUUCAUGCUACAGUACUG TP53BP2 CACCCAGAGAACAUUUAUUAAUAAAUGUUCUCUGGGUG CYBA UGGGGACAGAAGUACAUGA UCAUGUACUUCUGUCCCCA RAC1GAGUCCUGCAUCAUUUGAA UUCAAAUGAUGCAGGACUC SPP1 GUGCCAUACCAGUUAAACAUGUUUAACUGGUAUGGCAC SPP1 GCAAAAUGAAAGAGAACAU AUGUUCUCUUUCAUUUUGC ASPP1CGAACUCAGAGAAAUGUAA UUACAUUUCUCUGAGUUCG ASPP1 GGAGAAAAACGUACUGAAAUUUCAGUACGUUUUUCUCC SOX9 CCUUCAUGAAGAUGACCGA UCGGUCAUCUUCAUGAAGG

For all the above Structures (A)-(P), in various embodiments x=y andeach of x and y is 19, 20, 21, 22 or 23. In preferred embodiments,x=y=19. In additional embodiments the compound comprises modifiedribonucleotides in alternating positions wherein each N at the 5′ and 3′termini of (N)x are modified in their sugar residues and the middleribonucleotide is not modified, e.g. ribonucleotide in position 10 in a19-mer strand, position 11 in a 21 mer and position 12 in a 23-merstrand.

In some embodiments where x=y=21 or x=y=23 the position of modificationsin the 19 mer are adjusted for the 21 and 23 mers with the proviso thatthe middle nucleotide of the antisense strand is preferably notmodified.

For all the above Structures (A)-(P), in some embodiments, neither (N)xnor (N′)y are phosphorylated at the 3′ and 5′ termini. In otherembodiments either or both (N)x and (N′)y are phosphorylated at the 3′termini. In yet another embodiment, either or both (N)x and (N′)y arephosphorylated at the 3′ termini using non-cleavable phosphate groups.In yet another embodiment, either or both (N)x and (N′)y arephosphorylated at the terminal 2′ termini position using cleavable ornon-cleavable phosphate groups. These particular siRNA compounds arealso blunt ended and are non-phosphorylated at the termini; however,comparative experiments have shown that siRNA compounds phosphorylatedat one or both of the 3′-termini have similar activity in vivo comparedto the non-phosphorylated compounds.

For all the above Structures (A)-(P), in some embodiments, the compoundis blunt ended, for example wherein both Z and Z′ are absent. In analternative embodiment, the compound comprises at least one 3′ overhang,wherein at least one of Z or Z′ is present. Z and Z′ independentlycomprises one or more covalently linked modified or non-modifiednucleotides, for example inverted dT or dA; dT, LNA, mirror nucleotideand the like. In some embodiments each of Z and Z′ are independentlyselected from dT and dTdT. siRNA in which Z and/or Z′ is present havesimilar activity and stability as siRNA in which Z and Z′ are absent.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more locked nucleic acids (LNA) also definedas bridged nucleic acids or bicyclic nucleotides. Preferred lockednucleic acids are 2′-O, 4′-C-ethylene nucleosides (ENA) or 2′-O,4′-C-methylene nucleosides. Other examples of LNA and ENA nucleotidesare disclosed in WO 98/39352, WO 00/47599 and WO 99/14226, allincorporated herein by reference.

In certain embodiments for all the above-mentioned Structures, thecompound comprises one or more altritol monomers (nucleotides), alsodefined as 1,5 anhydro-2-deoxy-D-altrito-hexitol (see for example,Allart, et al., 1998. Nucleosides & Nucleotides 17:1523-1526; Herdewijnet al., 1999. Nucleosides & Nucleotides 18:1371-1376; Fisher et al.,2007, NAR 35(4):1064-1074; all incorporated herein by reference).

The present invention explicitly excludes compounds in which each of Nand/or N′ is a deoxyribonucleotide (D-A, D-C, D-G, D-T). In certainembodiments (N)x and (N′)y may comprise independently 1, 2, 3, 4, 5, 6,7, 8, or 9 deoxyribonucleotides. In certain embodiments the presentinvention provides a compound wherein each of N is an unmodifiedribonucleotide and the 3′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 consecutive nucleotides at the 3′ terminus of (N′)yare deoxyribonucleotides. In yet other embodiments each of N is anunmodified ribonucleotide and the 5′ terminal nucleotide or 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides at the 5′terminus of (N′)y are deoxyribonucleotides. In further embodiments the5′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, or 9 consecutivenucleotides at the 5′ terminus and 1, 2, 3, 4, 5, or 6 consecutivenucleotides at the 3′ termini of (N)x are deoxyribonucleotides and eachof N′ is an unmodified ribonucleotide. In yet further embodiments (N)xcomprises unmodified ribonucleotides and 1 or 2, 3 or 4 consecutivedeoxyribonucleotides independently at each of the 5′ and 3′ termini and1 or 2, 3, 4, 5 or 6 consecutive deoxyribonucleotides in internalpositions; and each of N′ is an unmodified ribonucleotide. In certainembodiments the 3′ terminal nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 13 or 14 consecutive nucleotides at the 3′ terminus of (N′)y andthe terminal 5′ nucleotide or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13 or14 consecutive nucleotides at the 5′ terminus of (N)x aredeoxyribonucleotides. The present invention excludes compounds in whicheach of N and/or N′ is a deoxyribonucleotide. In some embodiments the 5′terminal nucleotide of N or 2 or 3 consecutive of N and 1, 2, or 3 of N′is a deoxyribonucleotide. Certain examples of active DNA/RNA siRNAchimeras are disclosed in US patent publication 2005/0004064, andUi-Tei, 2008 (NAR 36(7):2136-2151) incorporated herein by reference intheir entirety.

Unless otherwise indicated, in preferred embodiments of the structuresdiscussed herein the covalent bond between each consecutive N and N′ isa phosphodiester bond.

An additional novel molecule provided by the present invention is anoligonucleotide comprising consecutive nucleotides wherein a firstsegment of such nucleotides encode a first inhibitory RNA molecule, asecond segment of such nucleotides encode a second inhibitory RNAmolecule, and a third segment of such nucleotides encode a thirdinhibitory RNA molecule. Each of the first, the second and the thirdsegment may comprise one strand of a double stranded RNA and the first,second and third segments may be joined together by a linker. Further,the oligonucleotide may comprise three double stranded segments joinedtogether by one or more linker.

Thus, one molecule provided by the present invention is anoligonucleotide comprising consecutive nucleotides which encode threeinhibitory RNA molecules; said oligonucleotide may possess a triplestranded structure, such that three double stranded arms are linkedtogether by one or more linker, such as any of the linkers presentedhereinabove. This molecule forms a “star”-like structure, and may alsobe referred to herein as RNAstar. Such structures are disclosed in PCTpatent publication WO 2007/091269, assigned to the assignee of thepresent invention and incorporated herein in its entirety by reference.

A covalent bond refers to an internucleotide linkage linking onenucleotide monomer to an adjacent nucleotide monomer. A covalent bondincludes for example, a phosphodiester bond, a phosphorothioate bond, aP-alkoxy bond, a P-carboxy bond and the like. The normal internucleosidelinkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. In certainpreferred embodiments a covalent bond is a phosphodiester bond. Covalentbond encompasses non-phosphorous-containing internucleoside linkages,such as those disclosed in WO 2004/041924 inter alia. Unless otherwiseindicated, in preferred embodiments of the structures discussed hereinthe covalent bond between each consecutive N and N′ is a phosphodiesterbond.

For all of the structures above, in some embodiments the oligonucleotidesequence of (N)x is fully complementary to the oligonucleotide sequenceof (N′)y. In other embodiments (N)x and (N′)y are substantiallycomplementary. In certain embodiments (N)x is fully complementary to atarget sequence. In other embodiments (N)x is substantiallycomplementary to a target sequence.

In some embodiments, neither (N)x nor (N′)y are phosphorylated at the 3′and 5′ termini. In other embodiments either or both (N)x and (N′)y arephosphorylated at the 3′ termini (3′ Pi). In yet another embodiment,either or both (N)x and (N′)y are phosphorylated at the 3′ termini withnon-cleavable phosphate groups. In yet another embodiment, either orboth (N)x and (N′)y are phosphorylated at the terminal 2′ terminiposition using cleavable or non-cleavable phosphate groups. Further, theinhibitory nucleic acid molecules of the present invention may compriseone or more gaps and/or one or more nicks and/or one or more mismatches.Without wishing to be bound by theory, gaps, nicks and mismatches havethe advantage of partially destabilizing the nucleic acid/siRNA, so thatit may be more easily processed by endogenous cellular machinery such asDICER, DROSHA or RISC into its inhibitory components.

In the context of the present invention, a gap in a nucleic acid refersto the absence of one or more internal nucleotides in one strand, whilea nick in a nucleic acid refers to the absence of an internucleotidelinkage between two adjacent nucleotides in one strand. Any of themolecules of the present invention may contain one or more gaps and/orone or more nicks.

The structures disclosed herein, when integrated into antisense andcorresponding sense nucleic acid sequences to any target gene, providessiRNA compound useful in reducing expression of that target gene. Thetarget gene is a mammalian or non-mammalian gene. The methods of theinvention comprise topically and non-invasively administering to the eyeof the subject one or more siRNA compounds which inhibit expression of atarget gene in the eye of the subject.

siRNA Synthesis

The compounds of the present invention can be synthesized by any of themethods that are well-known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotides. Such synthesis is, among others,described in Beaucage and Iyer, Tetrahedron 1992; 48:2223-2311; Beaucageand Iyer, Tetrahedron 1993; 49: 6123-6194 and Caruthers, et. al.,Methods Enzymol. 1987; 154: 287-313; the synthesis of thioates is, amongothers, described in Eckstein, Annu. Rev. Biochem. 1985; 54: 367-402,the synthesis of RNA molecules is described in Sproat, in Humana Press2005 edited by Herdewijn P.; Kap. 2: 17-31 and respective downstreamprocesses are, among others, described in Pingoud et. al., in IRL Press1989 edited by Oliver; Kap. 7: 183-208.

Other synthetic procedures are known in the art e.g. the procedures asdescribed in Usman et al., J. Am. Chem. Soc., 1987, 109:7845; Scaringeet al., NAR, 1990, 18:5433; Wincott et al., NAR 1995, 23:2677-2684; andWincott et al., Methods Mol. Bio., 1997, 74:59, and these procedures maymake use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Themodified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotidesare incorporated as desired.

The oligonucleotides of the present invention can be synthesizedseparately and joined together post-synthetically, for example, byligation (Moore et al., Science 1992, 256:9923; International PatentPublication No. WO 93/23569; Shabarova et al., NAR 1991, 19:4247; Bellonet al., Nucleosides & Nucleotides, 1997, 16:951; Bellon et al.,Bioconjugate Chem 1997, 8:204), or by hybridization following synthesisand/or deprotection.

It is noted that a commercially available machine (available, interalia, from Applied Biosystems) can be used; the oligonucleotides areprepared according to the sequences disclosed herein. Overlapping pairsof chemically synthesized fragments can be ligated using methods wellknown in the art (e.g., see U.S. Pat. No. 6,121,426). The strands aresynthesized separately and then are annealed to each other in the tube.Then, the double-stranded siRNAs are separated from the single-strandedoligonucleotides that were not annealed (e.g. because of the excess ofone of them) by HPLC. In relation to the siRNAs or siRNA fragments ofthe present invention, two or more such sequences can be synthesized andlinked together for use in the present invention.

The compounds of the invention can also be synthesized via tandemsynthesis methodology, as described for example in US Patent PublicationNo. 2004/0019001 (McSwiggen), and in PCT Patent Publication No. WO2007/091269 (assigned to the assignee of the present invention andincorporated herein in its entirety by reference) wherein both siRNAstrands are synthesized as a single contiguous oligonucleotide fragmentor strand separated by a cleavable linker which is subsequently cleavedto provide separate siRNA fragments or strands that hybridize and permitpurification of the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker.

The present invention further provides for a pharmaceutical compositioncomprising two or more siRNA molecules for the treatment of any of thediseases and conditions mentioned herein, whereby said two molecules maybe physically mixed together in the pharmaceutical composition inamounts which generate equal or otherwise beneficial activity, or may becovalently or non-covalently bound, or joined together by a nucleic acidlinker of a length ranging from 2-100, preferably 2-50 or 2-30nucleotides.

Thus, the siRNA molecules may be covalently or non-covalently bound orjoined by a linker to form a tandem siRNA compound. Such tandem siRNAcompounds comprising two siRNA sequences are typically about 38-150nucleotides in length, more preferably 38 or 40-60 nucleotides inlength, and longer accordingly if more than two siRNA sequences areincluded in the tandem molecule. A longer tandem compound comprised oftwo or more longer sequences which encode siRNA produced via internalcellular processing, e.g., long dsRNAs, is also envisaged, as is atandem molecule encoding two or more shRNAs. Such tandem molecules arealso considered to be a part of the present invention. A tandem compoundcomprising two or more siRNAs sequences of the invention is envisaged.

Additionally, the siRNA disclosed herein or any nucleic acid moleculecomprising or encoding such siRNA can be linked or bound (covalently ornon-covalently) to antibodies (including aptamer molecules) against cellsurface internalizable molecules expressed on the target cells, in orderto achieve enhanced targeting for treatment of the diseases disclosedherein. For example, anti-Fas antibody (preferably a neutralizingantibody) may be combined (covalently or non-covalently) with any of thesiRNA compounds.

The compounds of the present invention can be delivered either directlyor with viral or non-viral vectors. When delivered directly thesequences are generally rendered nuclease resistant. Alternatively thesequences can be incorporated into expression cassettes or constructssuch that the sequence is expressed in the cell as discussed hereinbelow. Generally the construct contains the proper regulatory sequenceor promoter to allow the sequence to be expressed in the targeted cell.Vectors optionally used for delivery of the compounds of the presentinvention are commercially available, and may be modified for thepurpose of delivery of the compounds of the present invention by methodsknown to one of skill in the art.

It is also envisaged that a long oligonucleotide (typically 25-500nucleotides in length) comprising one or more stem and loop structures,where stem regions comprise the sequences of the oligonucleotides of theinvention, may be delivered in a carrier, preferably a pharmaceuticallyacceptable carrier, and may be processed intracellularly by endogenouscellular complexes (e.g. by DROSHA and DICER as described above) toproduce one or more smaller double stranded oligonucleotides (siRNAs)which are oligonucleotides of the invention. This oligonucleotide can betermed a tandem shRNA construct. It is envisaged that this longoligonucleotide is a single stranded oligonucleotide comprising one ormore stem and loop structures, wherein each stem region comprises asense and corresponding antisense siRNA sequence of the genes of theinvention.

RNA Interference

A number of PCT applications have recently been published that relate tothe RNAi phenomenon. These include: PCT publication WO 00/44895; PCTpublication WO 00/49035; PCT publication WO 00/63364; PCT publication WO01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCTpublication WO 00/44914; PCT publication WO 01/29058; and PCTpublication WO 01/75164.

RNA interference (RNAi) is based on the ability of dsRNA species toenter a cytoplasmic protein complex, where it is then targeted to thecomplementary cellular RNA and specifically degrade it. The RNAinterference response features an endonuclease complex containing ansiRNA, commonly referred to as an RNA-induced silencing complex (RISC),which mediates cleavage of single-stranded RNA having a sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA may take place in the middle of the region complementaryto the antisense strand of the siRNA duplex (Elbashir et al., GenesDev., 2001, 15(2):188-200). In more detail, longer dsRNAs are digestedinto short (17-29 bp) dsRNA fragments (also referred to as shortinhibitory RNAs, “siRNAs”) by type III RNAses (DICER, DROSHA, etc.;Bernstein et al., Nature, 2001, 409(6818):363-6; Lee et al., Nature,2003, 425(6956):415-9). The RISC protein complex recognizes thesefragments and complementary mRNA. The whole process is culminated byendonuclease cleavage of target mRNA (McManus & Sharp, Nature Rev Genet,2002, 3(10):737-47; Paddison & Hannon, Curr Opin Mol Ther. 2003,5(3):217-24). (For additional information on these terms and proposedmechanisms, see for example Bernstein et al., RNA 2001, 7(11):1509-21;Nishikura, Cell 2001, 107(4):415-8 and PCT publication WO 01/36646).

Several groups have described the development of DNA-based vectorscapable of generating siRNA within cells. The method generally involvestranscription of short hairpin RNAs that are efficiently processed toform siRNAs within cells (Paddison et al. PNAS USA 2002, 99:1443-1448;Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS USA 2002,8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553). Thesereports describe methods to generate siRNAs capable of specificallytargeting numerous endogenously and exogenously expressed genes.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology used is intended to be in the natureof words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventioncan be practiced otherwise than as specifically described.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.The disclosures of these publications and patents and patentapplications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

The present invention is illustrated in detail below with reference toexamples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook etal., Molecular cloning: A laboratory manual, Cold Springs HarborLaboratory, New-York (1989, 1992), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1988), and as in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, APractical Guide to Molecular Cloning, John Wiley & Sons, New York(1988), and as in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein byreference. Polymerase chain reaction (PCR) was carried out as instandard PCR Protocols: A Guide To Methods And Applications, AcademicPress, San Diego, Calif. (1990). In situ PCR in combination with FlowCytometry (FACS) can be used for detection of cells containing specificDNA and mRNA sequences (Testoni et al., Blood 1996, 87:3822.) Methods ofperforming RT-PCR are well known in the art.

Cell Culture

HeLa cells (American Type Culture Collection) were cultured as describedin Czauderna, et al. (NAR, 2003. 31:670-82). Human keratinocytes werecultured at 37° C. in Dulbecco's modified Eagle medium (DMEM) containing10% FCS. The mouse cell line, B16V (American Type Culture Collection),was cultured at 37° C. in Dulbecco's modified Eagle medium (DMEM)containing 10% FCS. Culture conditions were as described in (MethodsFind Exp Clin Pharmacol. 1997, 19(4):231-9).

In each case, the cells were subject to the experiments as describedherein at a density of about 50,000 cells per well and thedouble-stranded nucleic acid according to the present invention wasadded at a concentration of 20 nM, whereby the double-stranded nucleicacid was complexed using 1 μg/ml of a proprietary lipid as describedbelow.

In the histochemical/microscopic figures, arrows were added to drawattention to staining of the tissue.

Animal Models Model Systems of Glaucoma and of Retinal Ganglion Cells(RGC) Death ONC Model in Rats

Various animal models are useful for studying the effect of siRNAtherapeutics in treating glaucoma. In the optic nerve crush model inrats the orbital optic nerve (ON) of anesthetized rats is exposedthrough a supraorbital approach, the meninges severed and all axons inthe ON transected by crushing with forceps for 10 seconds, 2 mm from thelamina cribrosa. Testing active inhibitors of the invention (such assiRNA) for treating or preventing glaucoma is performed, for example, inthe animal models described by Pease et al. (J. Glaucoma, 2006,15(6):512-9. Manometric calibration and comparison of TonoLab andTonoPen tonometers in rats with experimental glaucoma and in normalmice).

Optic nerve crush (ONC) model in adult Wistar rats is also an acceptedmodel for studying Retinal Ganglion Cells (RGC) death. The onset andkinetics of RGC death in this model are very reproducible; RGC apoptosisbegins on day 4-5 after the ONC; massive RGC loss (about 50-60%) isobserved on days 7-10 after the ONC; and 95% of the RGC loss is occursby week 3-4 after the ONC. This model allows for establishment of theneuroprotective efficacy of test drugs in vivo.

In some non-limiting examples, siRNA compounds directed to genes shownin Tables A1-A4 are tested in this animal model which show that thesesiRNA compounds treat and/or prevent glaucoma and/or RGC death whentopically and non-invasively delivered to the eye.

IOP Model in Rats

Intraocular pressure (IOP) is a measurement of the fluid pressure insidethe eye. This fluid, called aqueous humor, is circulated and thendrained out via specialized outflow pathways. If the drainage systemdoes not function properly, as in prevalent forms of glaucoma, pressureinside the eye builds up. A model of ocular hypertension in Brown Norwayrats developed by Dr. J. Morrison and collaborators at the Casey EyeInstitute (Portland, Oreg.) is used in this study. The Morrison modelinvolves injection of hypertonic saline into an episcleral vein, leadingto blockade of the aqueous humor outflow pathways. This procedure leadsto gradual increase of eye pressure and progressive death of RGCs.Importantly, inner retinal atrophy, optic nerve degeneration, and opticnerve head remodeling observed in this model are similar to that seen inhuman glaucoma. Thus, the Morrison model is considered the bestpre-clinical rodent model of glaucoma.

In Vivo Axotomy Model in Rats

In this model RGC apoptosis is induced by axotomy of the optic nerve(ON) in adult Sprague-Dawley rats. The onset and kinetics of RGC deathin this model system are very reproducible and allow for theestablishment of the neuroprotective efficacy of non-invasivelyadministered siRNA compound in vivo. Using this method, the time courseof RGC death follows a predictable course: cell death begins on day 5and proceeds to the rapid loss of more than 90% of these neurons by 2weeks.

Vehicle Formulations and Exemplary Eye Drop Formulations

The aqueous eye drop formulation optionally contain various additivesincorporated ordinarily, such as buffering agents (e.g., phosphatebuffers, borate buffers, citrate buffers, tartarate buffers, acetatebuffers, amino acids, sodium acetate, sodium citrate and the like),isotonicities (e.g., saccharides such as sorbitol, glucose and mannitol,polyhydric alcohols such as glycerin, concentrated glycerin,polyethylene glycol and propylene glycol, salts such as sodiumchloride), preservatives or antiseptics (e.g., benzalkonium chloride,benzethonium chloride, p-oxybenzoates such as methyl p-oxybenzoate orethyl p-oxybenzoate, benzyl alcohol, phenethyl alcohol, sorbic acid orits salts, thimerosal, chlorobutanol and the like), solubilizing aids orstabilizing agents (e.g., cyclodextrins and their derivative,water-soluble polymers such as polyvinyl pyrrolidone) surfactants suchas polysorbate 80 (Tween 80)), pH modifiers (e.g., hydrochloric acid,acetic acid, phosphoric acid, sodium hydroxide, potassium hydroxide,ammonium hydroxide and the like), chelating agents (e.g., sodiumedetate, sodium citrate, condensed sodium phosphate) and the like.

The eye drop formulation in the form of an aqueous suspension may alsocontain suspending agents (e.g., polyvinyl pyrrolidone, glycerinmonostearate) and dispersing agents (e.g., surfactants such as tyloxapoland polysorbate 80, ionic polymers such as sodium alginate), in additionto the additives listed above, thereby ensuring that the eye dropformulation is a further uniform microparticulate and satisfactorilydispersed aqueous suspension.

The ophthalmic ointment may comprise a known ointment base, such aspurified lanolin, petrolatum, plastibase, liquid paraffin, polyethyleneglycol and the like.

Exemplary Eye Drop Formulation 1:

Formulation of siRNA compounds in PBS (“naked siRNA formulation”) istypically prepared by dissolving dry siRNA in PBS. The formulationcomprises at least one siRNA compound typically present in an amountranging from about 5 μg/μl to about 60 μg/μl by volume of thecomposition.

In a non-limiting example formulation of an siRNA compound in PBS wasprepared as follows: Under sterile conditions, 500 mg of dry siRNA weredissolved in 25 ml of sterile double distilled water (DDW), to achieve aclear 20 mg/ml solution. The solution was stored at −80° C. until use.The 20 mg/ml stock solution in DDW was then brought to a workingconcentration of 100 μg/3 μl, in PBS, as follows: 325 μl of 20 mg/mlstock of siRNA solution (6.5 mg) were precipitates by 0.15M NaCl andEtOH, and dried under a tissue culture laminar (sterile conditions). 6.5mg of dry siRNA were dissolved in 130 μl PBS to form eye dropformulation 1.

Exemplary Eye Drop Formulation 2:

In some embodiments at least one siRNA compound is formulated intris(hydroxymethyl)aminomethane (TRIS) 1M, pH 8.0 (available e.g. fromSigma (catalog #T-1503)).

In a non-limiting example 121.1 g of TRIS base (Sigma #T1503) weredissolved in 700 ml of ddH₂O. Desired pH 8 was achieved with addition ofconcentrated HCl. DDW was added to have final 1 L solution. Understerile conditions, 42.426 mg of siRNA compound powder were dissolved in2.1 ml of sterile double distilled water, to achieve a clear 20 mg/ml(1.5 mM) stock solution. The stock solution was stored at −80° C. untiluse. Under sterile conditions, corresponding amounts of siRNA stock werelyophilized and re-suspended in corresponding amount of 1M TRIS pH 8.

Exemplary Eye Drop Formulation 3:

The concentration of the viscosity-enhancing agents to be used istypically within the range of about 0.05 to about 5.0% (w/v), and moredesirably, from about 0.1 to about 3.0% (w/v). One preferred ophthalmiccomposition comprises from about 0.01 to about 0.075% (w/v) of an activeingredient; from about 0.15 to about 2.5% (w/v) of polyoxyl 40 stearate;from about 0.1 to about 3.0% (w/v) of a cellulose thickening agentselected from 25 hydroxypropyl methylcellulose or hydroxyethylcellulose;and from about 0.0001% to about 0.01% (w/v) of an anti-oxidant selectedfrom butylated hydroxytoluene or sodium thiosulfate.

The combination of the surfactants such as polyoxyl 40 stearate orpolyoxyethylene cetyl ether or polyoxyethylene octylphenyl ether withsiRNA yields an eye-drop preparation causing less irritation to theeyes, providing better distribution in the eyes and having higherstability. The inclusion of an anti-oxidant and a cellulose-thickeningagent further improves the distribution of the active therapeutic agent(i.e. siRNA) in the eyes and the stability.

Other excipients may also be added, such as for example an isotonicagent, buffer, preservative, and or pH-controlling agent. Sterilepurified water in appropriate amounts is present to obtain the desiredeye-drop preparation.

The pH of the ophthalmic composition is within the range, which isnormally used for ophthalmic preparations, as known in the art, but isdesirably within the range of 5 to 8. The formulation further comprisesat least one siRNA compound typically present in an amount ranging fromabout 6.6 μg/μl to about 50 μg/μl by volume of the composition.

Exemplary Eye Drop Formulation 4:

In the topical delivery of drugs to the lens, drug retention on the eyesurface is considered to be important. Without wishing to be bound totheory, increased retention on the ocular surface leads to increasedocular absorption of a drug through the cornea into the aqueous humorand subsequently the lens.

In some embodiments a topical suspension is preferred. Non-limitingexamples of topical suspensions include:

(1) hydroxypropyl methylcellulose (HPMC, 0.5% w/v), (2) xanthan gum(0.5% w/v), (3) gellan gum (0.5% w/v), (4) carbopol (0.25% w/v), and (5)carbopol (0.25% w/v)-hydroxypropyl methylcellulose (HPMC) (0.25% w/v).Viscosity measurements are conducted with a viscometer.

The formulation further comprises at least one siRNA compound typicallypresent in an amount ranging from about 5 μg/μl to about 60 μg/μl byvolume of the composition.

Exemplary Eye Drop Formulation 5:

In ophthalmic compositions, a chelating agent is optionally used toenhance preservative effectiveness. Suitable chelating agents are thoseknown in the art, and, while not intending to be limiting, edetate(EDTA) salts like edetate disodium, edetate calcium disodium, edetatesodium, edetate trisodium, and edetate dipotassium are examples ofuseful chelating agents. It is understood that EDTA refers to a specieshaving four carboxylic acid functional groups, and that these carboxylicacid groups may be protonated or deprotonated (i.e. in the salt form)depending upon the pH of the composition it is in.

Buffers are commonly used to adjust the pH to a desirable range forophthalmic use. Generally, a pH of around 5-8 is desired, however, thismay need to be adjusted due to considerations such as the stability orsolubility of the therapeutically active agent or other excipients.

Another commonly used excipient in ophthalmic compositions is aviscosity-enhancing, or a thickening agent. Thickening agents are usedfor a variety of reasons, ranging from improving the form of theformulation for convenient administration to improving the contact withthe eye to improve bioavailability. The viscosity-enhancing agentcomprises a polymer containing hydrophilic groups such asmonosaccharides, polysaccharides, ethylene oxide groups, hydroxylgroups, carboxylic acids or other charged functional groups. While notintending to limit some examples useful viscosity-enhancing agents aresodium carboxymethylcellulose, hydroxypropylmethylcellulose, povidone,polyvinyl alcohol, and polyethylene glycol.

The formulation further comprises at least one siRNA compound typicallypresent in an amount ranging from about 5 μg/μl to about 60 μg/μl byvolume of the composition.

Exemplary Eye Drop Formulation 6 (“Formulation A”): Formulation “A”:Prepare 2 Solutions, A and B.

Preparation of Solution A: 4% solution of methylcellulose in water. 0.4g of methylcellulose 25 (ScienceLab.com, Cat# SLM2050) dissolved in thefinal volume of 10 ml of apyrogenic water for injections (WFI) in 50 mLsterile tube (Norbook, Cat#7082-51). Put small sterile stirrer insideand close the cap. Keep in boiling water bath while stirring for atleast 5 minutes until methylcellulose forms an opalescent solution.

Preparation of Solution B: 2% Glycerol; 0.02% (v/v) EDTA solution. Add333.3 uL of 60% glycerol (Sigma, Cat# G6279) & 2 μl 0.5M EDTA, pH=8(Sigma, Cat# E9884) (final—100 uM) to 9.665 ml WFI (Norbook,Cat#7082-51). Final volume—10 mL.

Prepare 2× (in regard to working concentration) solution of siRNA inSolution B. Note: if stock siRNA volume is substantially high, the finalvolume of Solution B is better kept lower than 10 mL to allow volumeadjustment later in 2× siRNA solution.

Preparation of Working siRNA Solution:

Cool solution A in hand ˜till 40-50° C. (still liquid) and mix thedesired volume with an equal volume of 2× solution of siRNA in SolutionB.

Final siRNA formulation contains 2% methylcellulose, 1% glycerol and0.01% (v/v) (50 uM) EDTA in WFI. Final pH should be ˜7.4 andosmomolarity—similar to human tear film. Concentration of siRNA in finalformulation is 33.3 mg/ml.

Must be prepared fresh once a week and aliquoted into the portions fordaily usage. The aliquots are kept at 4° C. Before application, the doseis warmed for 20-30 min at room temperature.

The final concentration of the methylcellulose is about 0.1% to about 3%w/v about 0.1% to about 2% w/v, about 0.1%-1.5% w/v methylcellulose,preferably about 2% w/v. The final concentration of glycerol is about0.1% to about 5% v/v, about 0.5% to about 2%, preferably about 1% v/v.The final concentration of EDTA is about 0.001% to about 0.05%, about0.005% to about 0.01%, preferably about 0.01% w/v.

Exemplary Eye Drop Formulation 7:

In some embodiments siRNA is formulated in a commercially availablelubricant eye drop formulation. Non-limiting example of commerciallyavailable lubricant eye drop formulation is Systane® available fromAlcon Inc. Such commercially available lubricant eye drop formulationstypically comprise polyethylene glycol and/or propylene glycol, anantiseptic and/or antiviral agent such as boric acid, a gelling agentsuch as hydroxypropyl guar, potassium chloride and/or sodium chlorideand/or magnesium chloride and/or calcium chloride and/or zinc chloride,preservatives such as Polyquad® and purified water. Under sterileconditions, 300 mg of siRNA powder is dissolved in 15 ml of steriledouble distilled water, to achieve a clear 20 mg/ml solution. Thesolution is stored at (−)80° C. until use. The 20 mg/ml stock solutionin double distilled water is then brought to a working concentration of100 μg/3 μl, in formulation, as follows:

To obtain a final formulated quantity of 1.7 mg siRNA: 85 μl of 20 mg/mlstock of siRNA (1.7 mg), is precipitated by 0.15M NaCl and EtOH, driedunder the tissue culture laminar (sterile conditions).

To achieve a siRNA solution of 33.3 mg/ml 1.7 mg dry siRNA is dissolvedin 51 μL Systane®.

siRNA Compounds Induce Knockdown (KD) of Target Genes

Example 1 In Vitro Testing of siRNA Compounds

About 1.5−2×10⁵ tested cells (HeLa cells and/or 293T cells for siRNAtargeting human genes and NRK52 (normal rat kidney proximal tubulecells) cells and/or NMuMG cells (mouse mammary epithelial cell line) forsiRNA targeting the rat/mouse gene) were seeded per well in 6 wellsplate (70-80% confluent).

About 24 hours later, cells were transfected with siRNA compounds usingthe Lipofectamine™ 2000 reagent (Invitrogen) at final concentrations of5 nM or 20 nM. The cells were incubated at 37° C. in a CO₂ incubator for72 h.

As positive control for transfection phosphatase and tensin homolog(PTEN)-Cy3 labeled siRNA compounds were used. Various chemicallymodified blunt ended siRNA compounds having alternating modified andunmodified ribonucleotides (modified at the 2′ position of the sugarresidue in both the antisense and the sense strands, wherein the moietyat the 2′ position is methoxy) and wherein the ribonucleotides at the 5′and 3′ termini of the antisense strand are modified in their sugarresidues, and the ribonucleotides at the 5′ and 3′ termini of the sensestrand are unmodified in their sugar residues were tested. Another siRNAcompound comprised a blunt ended structure having an antisense with analternating pattern of methoxy moieties and a sense strand with threeribonucleotides linked by two 2′S′ bridges at the 3′ terminus; andanother siRNA compound comprising antisense and sense strands havingthree ribonucleotides linked by 2′5′ bridges at the 3′ terminus wasused. Some of the tested compounds comprised a blunt ended structurehaving an antisense with an alternating pattern of methoxy moieties anda sense strand with one or two L-nucleotides at the 3′ terminal or 3′penultimate positions.

GFP siRNA compounds were used as negative control for siRNA activity. At72 h after transfection cells were harvested and RNA was extracted fromcells. Transfection efficiency was tested by fluorescent microscopy. Thepercent of inhibition of gene expression using specific preferred siRNAstructures was determined using qPCR analysis of a target gene in cellsexpressing the endogenous gene.

In general, the siRNAs having specific sequences that were selected forin vitro testing were specific for human and a second species such asnon-human primate, rat or rabbit genes. Similar results are obtainedusing siRNAs having these RNA sequences and modified as describedherein.

siRNAs targeted at genes related to apoptosis and neuroprotection weretested by a similar procedure and found active and able to induce knockdown of their corresponding target genes.

siRNA Prepared for Non-Invasive Administration and Administered by EyeDrops is Delivered to the Target Retinal Tissue in-vivo

Example 2 Distribution of CY3-Labeled siRNA in Murine Optic Tissue(siRNA Formulated in PBS) as Monitored by Fluorescence Microscopy andConfocal Microscopy

The current study showed siRNA delivery in-vivo to the lacrimal glandand ocular structures via a topical ocular route.

Abbreviations: E.D.=Eye Drops; ISH=In-situ Hybridization

The objective of this study was to test the delivery of DDIT4-Cy3labeled siRNA (siRNA compound targeting the RTP801 gene) to the lacrimalgland via non-invasive topical ocular route. The topical ocular routehas been evaluated for the delivery of siRNA into the lacrimal gland,anterior chamber of the eye, the retina and the optic nerve.

Materials and Methods

TABLE C1 Animals Species/strain: Mice/ICR, males Age: 6-10 weeks BodyWeight Range: 27-32 gr Group Size: 2 Total number of animals: 26

DDIT4-Cy3 siRNA (siRNA against mouse RTP801) formulated as 20 m-/mlstock solution in PBS and stored at −80° C. until use.

Experimental Procedure

The study included 6 experimental groups as described in Table C2hereinbelow: Mice were treated with a single siRNA DDIT4-Cy3 as follows:

Group V (A and B): dose regime: 50 μg/mouse/3 μl/eye, administrationroute: Right Eye drops (E.D.);

Group Am-V-A, Am3-VIII, Am3-IX: dose regime: 50 μg/mouse/3 μl/eye,administration route: both eyes, Eye drops (E.D.);

Group VI-A: dose regime: 20 μg/mouse/3 μl/eye, administration route:Right eye, Eye drops (E.D.);

Group VII, Am-VII: non-treated control.

TABLE C2 Study Design Injected Time SiRNA Dose Volume point Group GroupType μg/mouse (μl) Route (hrs) Size V-A DDIT4- 50.00 Right Eye 3.00 E.D.1 2 Cy3 Am-V-A DDIT4- 50.00 per eye/ 3.00 E.D. 1 2 Cy3 Both Eyes V-BDDIT4- 50.00 Right Eye 3.00 E.D. 4 2 Cy3 VI-A DDIT4- 20.00 Right Eye3.00 E.D. 1 2 Cy3 VII None none None none 5 Am-VII None none None none 1Am3-VIII DDIT4- 50.00 per eye/ 3.00 E.D. 4 2 Cy3 Both Eyes Am3-IX DDIT4-50.00 per eye/ 3.00 E.D. 24 2 Cy3 Both Eyes

The siRNA doses were prepared under sterile conditions. siRNA aliquotswere thawed for at least 30 min at room temperature prior toadministration. Total amount per aliquot (per each mouse) included anadditional 20% of the calculated volume. The designated siRNA dose wasdelivered in 3 μl (Experimental groups V-VI) volume per eye.

Anesthesia: Mice were anesthetized with Ketamine/Xylazine mix asfollows: 0.85 ml Ketamine+0.15 ml Xylazine+0.9 ml Saline, 0.1 ml mixsolution/20 gr body weight (BW).

Eye drop (E.D.) delivery: A 3 μl sample volume was slowly dropped in thetreated eye (corneal surface) using a blunt pipette tip; the animalswere placed in a warm environment to prevent anesthesia-inducedhypothermia, and were returned to cages after regaining consciousness.

Scheduled euthanasia: Following treatment, mice from all groups wereeuthanized according to the study design (Table C2, Time pointtermination).

Termination step was accomplished by cardiac puncture and bloodcollection; collected serum/plasma was stored (−20° C.) for furthersiRNA blood detection analysis.

Tissue Collection: Left and right eyes including optic nerve, lacrimalglands (left and right) from all animals were excised, brain (front andmiddle parts of the brain, frontal oriented sections) from animals ofgroups, V-A one from group VII, Am3-VIII, Am3-IX were excised, embeddedin optimal cutting temperature (OCT) compound and sectioned with acryostat into 8 μm slices that were placed on microscope slides(Superfrost/Plus™). Sections were fixed in 95% EtOH for 7 minutes thencounter stained with DAPI (1 μg/ml), incubated for 1 minute in absoluteEtOH, incubated for 5 minutes in xylene, air dried and mounted.

Tissue Evaluation: Delivery of siRNA was evaluated using lightmicroscopy and digital imaging. A tissue fragment was consideredpositive (i.e., successful Cy3 DDIT4 siRNA intracellular incorporation)only where histological (microscopic) examination showed a clearfluorescence signal within specific cells or structures such as acinaralone or in combination with ductal cells of the lacrimal gland.Background DAPI staining assisted in identification of tissue structure:in the case of anterior eye chamber structures such as cornea, angle,ciliary body or posterior eye chamber such as retina (retinal pigmentumepithelium cells (RPE), retinal ganglion cells (RGC)). Delivery wasconsidered positive if histological examination would show that allanimals within one experimental group showed the same fluorescencesignal within the investigated cell type/tissue. (i.e. time points orroute administration).

Results & Discussion

All cryosections were analyzed under light microscopy (bright field, BF)and fluorescence confocal microscopy. The fluorescent signal wasvisualized in the following tissues:

Retina by E.D. Delivery (Retinal Pigment Epithelial Cells, RetinalGanglion Cells)

FIGS. 1A-1B: Representative images of Cy3 labeled DDIT4 siRNAincorporated into mice retina.

FIG. 1A: Fluorescent microscopy (upper panel, magnification ×40) of theretina 1 hour after E.D. administration. Right arrow identifies Cy3stained retinal ganglion cells (RGC), left arrow identifies Cy3 stainedretinal pigmentum epithelium cells (RPE). Confocal microscopy (lowerpanel, magnification ×60) of the retinal ganglion cells layer. The lowerset of 9 views show Cy3 labeled tissue (Left), bright filed (BF, center)and a merge of the two (M, right). Arrows point to the labeled RGCs.

Figures in 1B: Confocal microscopy of the retina 4 hours after E.D.administration (upper ×40, lower ×60). Cy3 staining of the RGC isprominent.

Eye Drop Delivery of siRNA to the Lacrimal Glands (Acinar Cells, DuctalCells)

FIGS. 2A-2D: Representative images of Cy3 labeled DDIT4 siRNAincorporated into ductal and acinar cells of the murine lacrimal gland.

FIG. 2A: Fluorescent microscopy of the lacrimal gland 1 hour after E.D.administration (M=merged DAPI-Cy3 staining). Center arrow points toacinar cells, right arrow indicates ductal epithelial cells, the grayleft arrow points to the lacrimal duct (magnification ×60)

FIG. 2B: Confocal microscopy of the lacrimal gland 1 hour after E.Dadministration (upper ×40 and lower panel ×60). Arrow in merged image Mis pointing to lacrimal duct.

FIG. 2C: Confocal microscopy of the lacrimal gland 1 hour E.Dadministration (×40). Arrows are pointing to acinar cells

FIG. 2D: Confocal microscopy of the lacrimal gland 4 hours after E.D.(×60).

FIGS. 3A-3C show time course of accumulation of Cy3-siRNA in rat choroidafter administration by eye drops, following 1 and 4 hours postadministration. Choroid, outer nuclear layer, RPE and outer segmentlayer of photoreceptor cells show Cy3 staining.

FIG. 4 shows Cy3-siRNA delivery to the trabecular meshwork end ciliarybody one hour after administration by eye drops.

Different chemically modified siRNA (structural motives) were testedwith mouse Cy3-labeled siRNA. Similar tissue distribution to that shownabove was observed for all structures tested: Structure B havingalternating natural ribonucleotides and 2′O-Me sugar modifiedribonucleotides on both strands; Structure I having alternating naturalribonucleotides and 2′O-Me sugar modified ribonucleotides on theantisense strand and three ribonucleotides linked by 2′5′internucleotide linkages at the 3′ end of the sense strand; and astructure having three ribonucleotides linked by 2′5′ internucleotidelinkages at the 3′ end of each of the antisense and sense strands.

Example 3 Examination of Topical Ocular Route Delivery of FormulatedsiRNA in Rats (siRNA Formulated in PBS or in Methyl Cellulose 2%(“Formulation A”))

In the current study the ocular delivery of DDIT4-Cy3 siRNA formulatedeither in PBS or in Formulation A and applied by Eye Drops was studiedin rats.

TABLE C3 Study Design Route of Adm. Termi- Dose Adm. Volume nation GroupGroup siRNA μg/eye (Bilateral) (μl) Formulation time Size I DDIT4_1-Cy3100.00 E.D. 3.00 Formulated 30 min. 1 Ia DDIT4_1-Cy3 100.00 E.D. 3.00PBS 30 min. 1 II DDIT4_1-Cy3 100.00 E.D. 3.00 Formulated 1 hour 1 IIaDDIT4_1-Cy3 100.00 E.D. 3.00 PBS 1 hour 1 III DDIT4_1-Cy3 100.00 E.D.3.00 Formulated 3 hours 1 IIIa DDIT4_1-Cy3 100.00 E.D. 3.00 PBS 3 hours1 IV DDIT4_1-Cy3 100.00 E.D. 3.00 Formulated 6 hours 1 Iva DDIT4_1-Cy3100.00 E.D. 3.00 PBS 6 hours 1 V DDIT4_1-Cy3 100.00 E.D. 3.00 Formulated24 hours 1 Va DDIT4_1-Cy3 100.00 E.D. 3.00 PBS 24 hours 1 VI N/A N/AE.D. 3.00 vehicle 1 hours 1 VII N/A Intact N/A N/A N/A N/A N/A 1 control

Experimental Design:

1) Group I, II, III, IV, V: dose regime: siRNA formulated in FormulationA; 100 μg/rat/3 μl/eye, administration route: Bilateral Eye drops(E.D.);2) Group Ia, IIa, IIIa, IVa, Va: dose regime: siRNA formulated in PBS;100 μg/rat/3 μl/eye, administration route: Bilateral Eye drops (E.D.);3) Group VI: control group treated with the formulation vehicle ofFormulation A (no siRNA); 100 μg/rat/3 μl/eye, administration route:Bilateral Eye drops (E.D.);4) Group VII: intact control

The siRNA doses were prepared under sterile conditions. siRNA aliquotswere thawed for at least 30 minutes at room temperature prior toadministration. Total amount per aliquot (per each mouse) included anadditional 20% of the calculated volume. The designated siRNA dose wasdelivered in 3 μl volume per eye.

Anesthesia: Rats from groups I-VII were anesthetized with Equithesine (4ml/kg)

Termination step: At study termination, animals were deeply anesthetizedby Equithesine (4 ml/kg). Thereafter, rats were intracortically perfusedwith fresh 10% neutral buffered formalin using peristaltic pump standardregime.

Tissue Collection: Left and right eyes including optic nerve from allanimals were enucleated, perforated and post fixed in 10% neutralbuffered formalin for an additional 1 hour in room temperature with slowrotation, cryoprotected step-wise by sucrose gradient at 4° C.overnight, following cryoprotection in optimal cutting temperature (OCT)compound at 4° C. overnight with rotation, embedded in OCT compound andsectioned with a cryostat into 12 μm slices that were placed onmicroscope slides (Superfrost/Plus). Sections were counter stained withDAPI (1 μg/ml) and mounted.

Delivery of siRNA was evaluated using light microscopy and digitalimaging. A tissue fragment was considered positive (i.e., a successfulCy3 DDIT4_(—)1 siRNA transfer in anterior eye chamber, retina and opticnerve delivery was considered positive) if histological examinationwould show that all animals within one experimental group showed thesame fluorescent signal within the specific tissue/cell type.

Results: All cryosections were analyzed under light microscopy (brightfield, BF) and fluorescence confocal microscopy. The fluorescent signalwas visualized in retinal pigment epithelial cells and retinal ganglioncells. Notably, compounds formulated in PBS reached maximum distributionin ocular tissues 3 hours after administration, and were cleared fromsaid tissues at the 6 hour time point. However, compounds formulated inFormulation A were maximally distributed in ocular tissues at 24 hoursafter administration.

Example 4 Ocular Distribution of siRNA (Formulated in PBS) in theCynomolgus Monkey

TABLE C4 Experimental Design Termina- Processing tion after at termina-Animal Dose adminis- tion Group number Compound Volume tration Eyes 1101 Cy3-DDIT4_1 500 ug/20 1 hour 4% PFA ul/eye 2 251 DDIT4_1 500 ug/20 1hour Snap frozen ul/eye in liquid nitrogen 3 351 Cy3-DDIT4_1 500 ug/20 5hours 4% PFA ul/eye 4 401 DDIT4_1 500 ug/20 5 hours Snap frozen ul/eyein liquid nitrogen ED = Eye Drops, topically

The aim of this study was to investigate the tissue distribution ofsiRNA formulated in PBS following single topical ocular administrationto the surface of the eye of the Cynomolgus Macaca fascicularis monkey.All tissues were snap frozen in triplicate ˜1 to ˜2 g samples or as awhole, as appropriate.

RNA Purification

Frozen samples were grinded to a fine powder under liquid nitrogen. Asmall amount of the powdered tissue was used for RNA extraction. Therest of the tissues were stored at −80° C. for further usage. PolyA RNAwas extracted from each sample with MicroPoly(A)Purist mRNA purificationKit (#1919) and processed according to manufacturer's protocol forpoly(A)RNA isolation from tissues or cells. The final yields andspectral characteristics of the RNA are summarized below in Table C5.

TABLE C5 polyA RNA from monkey Concentration Tissue 260/280 260/230μg/μl Yield μg Lymph Node 1.58 0.92 0.104 1.55 Spleen 1.74 1.27 0.1752.62

One μg from the spleen and 0.7 μg from the lymph node polyA RNApreparations were used for cDNA synthesis using random primer. Casp2gene was successfully amplified from both cDNA preparations.

Results are summarized below in Table C6.

TABLE C6 Results Tissue Termi- Treatment analysed nation Av SD Groupsize ED(REDD14) Neuronal 1 h 0.2 [+/−0] 1 (2 eyes) Retina ED(REDD14)Neuronal 5 h 0.05 [+/−0.03] 1 (2 eyes) Retina ED(REDD14) RPE 1 h 0.62[+/−0.05] 1 (2 eyes) ED(REDD14) RPE 5 h 0.1 [+/−0.07] 1 (2 eyes)ED(REDD14) optic nerve 1 h 8.9 [+/−2.6] 1 (2 eyes) ED(REDD14): opticnerve 5 h 3 [+/−1.9] 1 (2 eyes)

Conclusion: SiRNA formulated in PBS following single topical ocularadministration to the surface of the eye of the Cynomolgus Macacafascicularis monkey reaches the neuronal retina and can be measured bythe qPCR S&L method.

Example 5 Delivery of siRNA (Formulated in TRIS) to Target RetinalTissue in Mice

Examination of topical delivery of siRNA molecules labeled withdifferent fluorophores.

Topical delivery to Mice of 20 ug of different fluorescently-labeledsiRNAs formulated in tris(hydroxymethyl)aminomethane (TRIS) 1M, pH 8, 1hr post-administration.

siRNAs tested:

Cy3-QM5; Cy3-DDIT4; Cy3.5-DDIT4; DDIT4_(—)1 Dy-649/C6; FITC-CNL_(—)1;3′Cy3-CNL_(—)1; Cy3-Casp2_(—)4 L-DNA; Cy3-AS-Casp2_(—)4; TGASEII-FAM;HNOEL-FAM. (QM5 is a rat/mouse siRNA that targets p53; CNL is controlscrambled siRNA)

Exemplary chemically modified Casp2 siRNA compounds were as follows:

1. Sense: GCCAGAAUGUGGAACUC2pC2pU, 17 and 18 are 2′-5′-bridge

-   -   Antisense: mAGmGAmGUmUCmCAmCAmUUmCUmGGmC-cy3

2. Sense: GCCAGAAUGUGGAACUC; LdC; LdT 18 and 19 are L-DNA

-   -   Antisense: mAGmGAmGUmUCmCAmCAmUUmCUmGGmC-cy3        3. Sense: GCCAGAAUGUGGAACUC; LdC; U18 is an L-DNA moiety    -   Antisense: mAGmGAmGUmUCmCAmCAmUUmCUmGGmC-cy3

LdC, LdT refer to L-DNA nucleotides, mA, mC, Mg, Mu refer to 2′O-methoxyribonucleotides.

Formulated (formulated compound): siRNA in 1M Tris pH 8.0 Of thefollowing siRNA's: QM5-Cy3#1, QM5-Cy3#10, DDIT4_(—)1-Cy3,DDIT4_(—)1Cy3.5, Redd14(DDIT4_(—)1) Dy-649/C6, FITC-CNL_(—)1RD/CNL_(—)1FD, scarmbled3′Cy3CNL 1, Cy3-AS-CASP2_(—)4-Struc-L-DNA-s (2residues at 3′ of sense)-plus-alt AS, Cy3AS-CASP2_(—)4-Struc2-5-s (2residues at 3′ of sense)-plus-alt AS, TGASEII-FAM, HNOEL-FAM.

TABLE C7 Formulations siRNA siRNA IDO Final Stock Preparation QM5-Cy3#1116937 40 μg 20 mg/ml 2 μl stock were lyophilized and resuspended in 7μl 1M Tris pH 8 QM5-Cy3#10 116938 40 μg 20 mg/ml 2 μl stock werelyophilized and resuspended in 7 μl 1M Tris pH 8 DDIT4_1-Cy3 117821 40μg 20 mg/ml 2 ul stock were lyophilized and resuspended in 7 μl 1M TrispH 8 DDIT4_1-Cy3.5 112206 40 μg 20 mg/ml 2 ul stock were lyophilized andresuspended in 7 μl 1M Tris pH 8 Redd14(DDIT4_1) 114029 40 μg 20 mg/ml 2μl stock were lyophilized and Dy-649/C6 resuspended in 7 μl 1M Tris pH 8FITC- 110357 40 μg 20 mg/ml 2 μl stock were lyophilized andCNL_1RD/CNL_1 resuspended in 7 μl 1M Tris pH 8 FD scarmbled3′Cy3- 11091040 μg 20 mg/ml 2 μl stock were lyophilized and CNL_1 resuspended in 7 μl1M Tris pH 8 Cy3-AS-CASP2_4- 122994 40 μg 20 mg/ml 2 μl stock werelyophilized and Struc-L-DNA-s(2 resuspended in 7 μl 1M Tris pH 8residues at 3′ of sense)-plus-alt AS Cy3-AS-CASP2_4- 122979 40 μg 20mg/ml 2 μl stock were lyophilized and Struc2-5-s(2 resuspended in 7 μl1M Tris pH 8 residues at 3′ of sense)-plus-alt AS TGASEII-FAM 7575511.62 μg 0.75 μg/μl 15.5 μl stock were lyophilized and resuspended in 2μl 1M Tris pH 8 HNOEL-FAM 75757 4.48 μg 1.4 μg/μl 3.2 μl stock werelyophilized and resuspended in 0.784 μl 1M Tris pH 8 DDIT4_1-Cy3 11782140 μg 20 mg/ml 2 μl stock + 0.7 ul PBSx 120 + 4.3 μl DDW

Description of the test material: Under sterile conditions,corresponding amount of siRNA stock were lyophilized and re-suspended incorresponding amount of 1M Tris pH8 (see Table C7).

Quantity supplied: One vile for each tested siRNA (Synthesized byBiospring, AG)

Storage Conditions: frozen until use. Prior to use, samples were thawedand kept at room temperature for 30 minutes.

Control Article(S)

Positive Control—DDIT4_(—)1-Cy3 in PBS, Batch #: 117821

Description of the test material: Under sterile conditions, 42.426 mg ofDDIT4_(—)1-Cy3 powder (BioSpring) were dissolved in 2.1 ml of steriledouble distilled water, to achieve a clear 20 mg/ml (1.5 mM) solution.The solution was stored at −80° C. until use.

Vehicle—1M Tris pH 8.0—Outsourcing from Sigma (catalog #T-1503). 121.1 gof TRIS base (Sigma # T1503) were dissolved in 700 ml of ddH₂O. DesirepH 8 was achieved with concentrated HCl. DDW was added to have final 1 Lsolution.

Vehicle—PBSX10—Outsourcing from Biological Industries (catalog#02-023-5A; (For 10×PBS) Batch #:619113)

Test System

Animals:

Species: Mice; Strain: RTP-801WT; CMF-608WT

Source: Harlan Laboratories, Jerusalem, Israel.

Age: 8-12 weeks; Body Weight Range: 17-28 gr

Sex: males; Group Size: 1; Total number of animals: 14

Animal Husbandry: Diet: Animals were provided an ad libitum commercialrodent diet and free access to drinking water. Environment: (i)Acclimatization of at least 5 days. (ii) All the animals were confinedin a limited access facility with environmentally-controlled housingconditions throughout the entire study period, and maintained inaccordance with approved standard operating procedures (SOPs).

Experimental Design

General: The study included 14 experimental groups as described in TableC8:

Experimental groups 1-12 siRNA delivered by eye drops, group 13 thevehicle [TRIS 1M pH8 treated control delivered by eye drops and group 14(non-treated control). Mice were treated with a single siRNA as follows:

-   -   Groups 1-9 and 12: dose regime: 20 μg/eye/3 μl/SiRNA, group 10:        dose regime 5.81 μg/eye/3 μl/siRNA, group 11: dose regime 2.24        μg/eye/3 μl/siRNA administration route: Eye Drop (E.D.);    -   Group 13: vehicle (TRIS 1M pH8) 3 μl/eye control    -   Group 14: none treated control

TABLE C8 Study Design Time Dose Volume point Group Group SiRNAtype/bilateral μg/eye (μl) Route (hrs) Size 1 QM5-Cy3#1 20 3.00 E.D. 1 12 QM5-Cy3#10 20 3.00 E.D. 1 1 3 DDIT4_1-Cy3 20 3.00 E.D. 1 1 4DDIT4_1-Cy3.5 20 3.00 E.D. 1 1 5 DDIT4_1 Dy-649/C6 20 3.00 E.D. 1 1 6FITC-CNL_1RD/CNL_1FD 20 3.00 E.D. 1 1 7 scarmbled3′cy3-CNL_1 20 3.00E.D. 1 1 8 Cy3-AS-CASP2_4-Struc-L-DNA-s(2 20 3.00 E.D. 1 1 residues at3′ of sense)-plus-alt AS 9 CY3-AS-CASP2_4-Struc2-5-s(2 20 3.00 E.D. 1 1residues at 3′ of sense)-plus-alt AS 10 TGASEII-FAM 5.81 3.00 E.D. 1 111 HNOEL-FAM 2.24 3.00 E.D. 1 1 12 DDIT4_1-Cy3 in PBS vehicle (positive20 3.00 E.D. 1 1 control) 13 Vehicle (TRIS 1M pH 8) (negative N/A 3.00E.D. 1 1 control) 14 Non treated (negative control) N/A 3.00 E.D. 1 1

The siRNA doses for delivery were prepared under sterile conditions andstored at −20° C. siRNA aliquots were thawed for at least 30 minutes atroom temperature prior to administration. Total amount per aliquot (pereach eye) included an additional 20% of the calculated volume. Thedesignated siRNA dose was delivered in 3.5 μl (Experimental groups 1 to9 and 12) volume per eye. The designated siRNA dose was delivered in 3.4μl (Experimental groups 10 and 11) volume per eye.

Anesthesia: Mice were anesthetized with Ketamine/Xylazine mixture asfollows (0.85 ml Ketamine+0.15 ml Xylazine+0.9 ml Saline, 0.1 ml mixsolution/20gr BW).

Eye drop delivery: A 3 μl volume sample was slowly dropped in each eye(corneal surface) by blunt pipette tip; the animals were placed in awarm environment to prevent anesthesia-induced hypothermia, and werereturned to its cage after it regaining consciousness.

Scheduled euthanasia: Mice from all groups were terminated according tothe study design (Table C8, Time points termination).

Termination step was accomplished by cervical dislocation.

Tissue Collection: Left and right eyes including optic nerve, from allanimals were enucleated, the retina was transversal dissected with theblade, the lens/vitreous were gently removed. The remaining eye cup wasfixed with 4% PFA (in PBS pH 7.4) for 30 minutes, then infiltrated with30% sucrose for 3 hours at 4° C. Washed 3×5 minutes with ice cold PBSpH7.4. It was then embedded in optimal cutting temperature (OCT)compound and sectioned longitudinal with a cryostat into 4 μm slicesthat were placed on microscope slides (Superfrost/Plus). Sections werecounter stained with DAPI (1 μg/ml) and mounted.

A whole blood drop from all animals, was smeared on the slide with thecover slip, and covered. It was then glued with nail polish

Evaluation

Delivery of siRNA was evaluated using light microscopy and digitalimaging. A tissue fragment was considered positive (i.e., a successfulsiRNA transfer\ intracellular incorporation occurred) only ifhistological (microscopic) examination showed clear fluorescence signalwithin specific cells or structures of the anterior or posteriorchamber, and retina. Background DAPI staining was assisted inidentification of tissue structure. Delivery was considered positive ifhistological examination was consistent within the group (bilateralidentical cell type or structural staining), i.e. the histologicalexamination would show the same fluorescent signal in all celltypes/tissues (anterior eye chamber, retina and optic nerve) of the sameexperimental group.

Results

FIGS. 5A-5B: Confocal microscopy (magnification ×60) of the retina 1hour after eye drop administration of QM5—siRNA targeted at the p53gene. SiRNA in retina (retinal pigment epithelial cells, retinalganglion cells) is shown by Cy3 fluorescence.

FIGS. 6A-6B are representative images of Cy3 labeled DDIT4 siRNAincorporated into mice retina.

FIG. 6B shows accumulation of DDIT4_(—)1 Cy3-siRNA 1 hour postadministration by eye drops. Choroid, outer nuclear layer, RPE and outersegment layer of photoreceptor cells show Cy3 staining.

FIG. 7 shows accumulation of DDIT4_(—)1 Dy-649/C6-siRNA 1 hour postadministration by eye drops in RGC cells by use of Dy-649/C6 staining.

FIG. 8A-8C represent control siRNA FITC-CNL_(—)1RD/CNL_(—)1FD andscarmbled3′ cy3-CNL_(—)1 delivery to retinal tissues by differentstaining methods. FIGS. 9A and 9B show delivery of different structuresof Casp2 to the mouse retinal tissues.

FIGS. 10A and 10B show TGASEII-FAM and HNOEL-FAM delivery.

FIG. 11 shows retinal delivery of siRNA against p53 in PBS as positivecontrol group and

FIGS. 12A and 12B show that in intact animals or when administering EDwithout siRNA no fluorescent signal is obtained in the retina.

Conclusion: No sequence dependent differences, neither fluorophore norgene dependent differences in the delivery to the retina were found. Thestudy showed efficient delivery of Cy3 conjugated siRNA compoundsdirected to CASP2, RTP801, TIGASEII and p53 target genes, to ocularstructures: RGC, RPE, photoreceptors, choroid.

Example 6 Quantification of Delivery of Casp2 siRNA (Formulated in PBSor in MC 2% (Formulation “A”)) to Target Retinal Tissue in Rats In-Vivo

Examination of different eye Drop formulations for the delivery of siRNAto the retina. The objective of the study was the determination ofCASP2_(—)4_S510 siRNA quantity in rat retina at time points 1, 3, 6 & 24hours following single topical administration in different eye drops(ED) formulations.

Test Article

Substance (unformulated compound): CASP2_(—)4_S510 (siRNA Against CASP2)

Supplied by Agilent (Manufacturer's catalog #QPI-1007, Batch #:Q02F08002N)

Description of the test material: CASP2_(—)4_S510: S— inverted-Abasic5′-cap, L-DNA 18/AS-AL. A 19-mer chemically modified blunt-ended duplexhaving two separate strands, with a sense strand (SEN) comprisingunmodified ribonucleotides (upper case letters), anL-deoxyribonucleotide at position 18 (bold, underlined) and inverteddeoxyabasic moiety (iB) present at the 5′ terminus of the SEN strand;and with an antisense strand (AS) comprising unmodified ribonucleotides(upper case letters), and 2′OMe sugar modified ribonucleotides (lowercase letters) at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 as shown inFormula I:

Formula I SEN 5′ iB-GCCAGAAUGUGGAACUCCU 3′ AS 3′ cGgUcUuAcACcUuGaGgA 5′

Storage Conditions: −80° C.

siRNA formulated in PBS: 33.3 mg/ml CASP2_(—)4_S510 in PBS (solution foreye drops)

Description of the test material: Under sterile conditions, 300 mg dryCASP2_(—)4_S510 siRNA were dissolved in 15 ml of sterile doubledistilled water, to achieve a clear 20 mg/ml solution. The solution wasstored at −80° C. until use. The 20 mg/ml stock solution in DDW was thenbrought to a working concentration of 100 μg/3 μl, in PBS, as follows:

6.5 mg CASP2_(—)4_S510 siRNA: 325 μl of 20 mg/ml stock of CASP2_(—)4(6.5 mg), were precipitated by 0.15M NaCl and EtOH, and dried under thetissue culture laminar (sterile conditions). siRNA solution 33.3 mg/ml:6.5 mg dry siRNA were dissolved in 195 μl PBS×1 Quantity prepared: 6.5mg/195 μl aliquoted into 4 tubes. Storage Conditions: freshly prepared

siRNA formulated in Formulation A: 33.3 mg/ml solution ofCASP2_(—)4_S510 siRNA in 2% (w/v) methylcellulose & 1% (v/v) sterileglycerol & 0.01% (w/v) EDTA solution in pyrogen free water (solution foreye drops)

Description of the test material: Under sterile conditions, 300 mg dryCASP2_(—)4_S510 siRNA were dissolved in 15 ml of sterile doubledistilled water, to achieve a clear 20 mg/ml solution. The solution wasstored at −80° C. until use. The 20 mg/ml stock solution in DDW wasbrought to a working concentration of 100 μg/3 μl, in formulation, asfollows:

6.5 mg CASP2_(—)4_S510 siRNA: 325 μl of 20 mg/ml stock of CASP2_(—)4(6.5 mg), were precipitated by 0.15M NaCl and EtOH, and dried under thetissue culture laminar (sterile conditions).

siRNA solution 33.3 mg/ml: 6.5 mg dry siRNA were dissolved in 195 μlFormulation A solution

Quantity prepared: 6.5 mg/19411 aliquoted into 4 tubes

Storage Conditions: freshly prepared

Control Article(s)

Methyl cellulose formulation (no siRNA)-2% methylcellulose & 1% v/vsterile glycerol & 0.01% w/v EDTA solution in pyrogen free water, wasprepared as follows:

Solution A: 0.4 g of methylcellulose 25 (ScienceLab.com, Cat# SLM2050)dissolved in final volume of 10 ml of hot boiled water (80-90° C.),(Norbrook), cooled down to room temperature and added in a proportion of1:1 (final concentration 2%) to solution B.

Solution B: 332 pd of 60% glycerol (Sigma, Cat# G6279) & 2 pd EDTAsolution pH8 (prepared from Sigma, Cat# E9884) in 9.66 ml WFI(Norbrook).

500 μL of Solution A mixed with 500 μL of Solution B to obtain: 2%methylcellulose & 1% v/v sterile Glycerol & 0.01% w/v EDTA solution inpyrogen free water (final pH was approximately 7.4 and osmolaritysimilar to human tear film).

PBS was supplied by Biological industries (Manufacturer catalog#02-023-5A (For 10×PBS); Batch #619113)

Test System

Animals used: Species: Rats; Strain: Adult, Sprague-Dawley (SD)

Source: Harlan, Jerusalem Israel

Age: 8-10 weeks; Body Weight Range: 180-250 gr

Sex: Male; Group Size: n=6/3; Total number of animals: 54

Animal Husbandry: Diet: Animals were provided an ad libitum commercialrodent diet and free access to drinking water.

Environment:

(i) Acclimatization of at least 5 days.

(ii) All animals were confined in a limited access facility withenvironmentally-controlled housing conditions throughout the entirestudy period, and maintained in accordance with approved standardoperating procedures (SOPs). Animals were provided ad libitum acommercial rodent diet (Harlan Teklad 2018S Global 18% Protein RodentDiet) and filtered, chlorinated and acidified water.

Rats were kept in microisolator cages with filter top, 1-6 Rats/cage.The cages were maintained under controlled environmental—conditions oftemperature (20-24° C.), relative humidity (30-70%), a 12-hr light/12-hrdark cycle, monitored by the control computer, throughout the studyperiod.

Experimental Design

Study design: Retinal concentration of CASP2_(—)4 siRNA following EDapplication of ocular formulation was determined by qPCR in study groupsterminated at different time points after siRNA administration. Eachexperimental siRNA-treated group included 6 rats. ED was appliedbilaterally as detailed in Study Design Table C9.

Termination time points post siRNA administration: Termination setup wasperformed according to the study design Table C9. Dosing and terminationof experimental groups were performed on separate days.

TABLE C9 Study Design Ter- Method Dose of Bilateral mina- of Group GroupED [100 μg/eye/ tion Anal- Number Size Test Article 3 ul] [hrs] ysis 1 6CASP2_4_S510 PBS formulated 1 qPCR siRNA 2 6 CASP2_4_S510 MC formulated1 qPCR 3 6 CASP2_4_S510 PBS formulated 3 qPCR siRNA 4 6 CASP2_4_S510 MCformulated 3 qPCR 5 6 CASP2_4_S510 PBS formulated 6 qPCR siRNA 6 6CASP2_4_S510 VIC formulated 6 qPCR 7 6 CASP2_4_S510 PBS formulated 24qPCR siRNA 8 6 CASP2_4_S510 VIC formulated 24 qPCR 9 3 N/A Vehicle MC 1qPCR 10 3 N/A intact N/A qPCR

Anesthesia: In the course of the experiment, the animals wereanesthetized with Equithesine (I.P. 4 ml/kg) for ED application and/orbefore terminations.

Eye Drop Delivery: A 3 μl sample volume of the test article or vehiclewas applied to the corneal surface bilaterally to the anesthetizedanimals, by a blunt pipette tip (filter tips 10 μl sterile (short)). Theanimals were placed in a warm environment to prevent anesthesia-inducedhypothermia, and were returned to their cage after regainingconsciousness (experimental Groups 7 and 8). Termination of animals fromall study groups was according to study design. Tissues were collectedaccording to study design.

Scheduled euthanasia: All animals were deeply anaesthetized andeuthanized according to the study design (Table C9, Termination).

Perfusion setup: Rats were perfused transcardially with PBS (20-50ml/min following 2-3 min).

Tissue Collection: After perfusion, both eyes (left and right) wereenucleated and stored on ice. The eyes were dissected using amicroscope, and optionally gross pathology was graded according to thesample grading scale. The cornea was dissected by a cut along thelimbus, lens was gently removed, and the retina and vitreous werecarefully separated from the sclera. Whole retinas and vitreous bodies(humor) were collected (Retina including: “neural retina”+RetinalPigment Epithelium+Choroid) into two separate appropriate and properlymarked test tubes. Dissected retinas were washed in a large volume ofPBS (each retina in a separate tube with fresh PBS), extra liquid wasremoved with Kimwipe® and retinas were snap-frozen in liquid nitrogen.Retina and Vitreous body samples were s subjected to RNA extraction.

Evaluation

RNA Extraction:

Retina: RNA was extracted from each retina sample individually (left andright) by double extraction. The RNA was transferred for cDNApreparation and qPCR analyses.

Vitreous: Material for Casp2_(—)4 siRNA detection from vitreous wasobtained using the following protocol:

Each rat vitreous was around 10 μl. 500 μl of reagent A EZ-RNA II(Biological Industries Cat no. 20-410-100) were added to each vitreous.The sample was homogenized, and 10 μg tRNA (1 μl from 10 mg/ml stock)were added. The sample was stored for 5 minutes at room temperature.Subsequently: EZ-RNA II B: 400 μl and EZ-RNA II C: 90 μl were added andMixed well. The sample was stored for 10 min at room temperature thencentrifuged 12000 g for 15 min at 4° C.

The upper phase was transferred to a fresh tube to which isopropanol:500 μl and Linear Acrylamide: 5 μl were added. The sample was storedovernight at −20° C., centrifuged at 12000 g for 20 min at 4° C., washedtwice with 75% ethanol. The pellet was dissolved in 15 μl H₂O.

siRNA Quantification: The quantity of the siRNA in retinas and vitreoushumor (siRNA quantification was performed per vitreous) was examined byqPCR siRNA quantification. qPCR was performed according to Quark'sstandard operating procedures. CASP2_(—)4 siRNA and reference geneexpression were tested. Results are summarized in Table C10.

TABLE C10 Results fmol siRNA/μg Termination Formulation Eye Group sizeretinal RNA 1 hrs Methyl Right 5 12.50 Cellulose Left 6 10.19 PBS Right6 8.08 Left 6 4.77 3 hrs Methyl Right 5 1.76 Cellulose Left 5 2.47 PBSRight 6 6.48 Left 6 7.80 6 hrs Methyl Right 4 1.37 Cellulose Left 6 3.80PBS Right 6 3.61 Left 6 1.04 24 hrs  Methyl Right 6 0.11 Cellulose Left6 0.21 PBS Right 6 0.03 Left 6 0.04

Conclusion: The study showed efficient delivery of siRNA into retina 1and 3 hours post administration. qPCR analysis showed that afterapplication of 100 μg siRNA in Formulation A, in average 11 fMol/ug oftotal RNA was obtained in the retina after 1 hour and 2 fMol/ug siRNAafter 3 hours. When administering the same concentration in PBS, anamount of 6 fmol/μg of siRNA was obtained after 1 hour and 8 fmol/μg oftot RNA was obtained after 3 hours.

Example 7 Quantification of Non-Invasively Administered siRNA Compound(Formulated in Various Formulations) in Target Retinal Tissue in Rats

Examination of different eye drop formulations for non-invasive deliveryof siRNA to the retina in vivo.

The objective of this study was the assessment of CASP2_(—)4_S510 siRNAquantity in normal rat retina at 3 hours following single topicalapplication of different siRNA formulations prepared for administrationas eye drops (ED).

Test Article

Substance (unformulated compound) CASP2_(—)4_S510 (siRNA Against CASP2)Supplied by Agilent (Manufacturer's catalog #QPI-1007; Batch #:Q02F08002N).

Description of the test material: CASP2_(—)4_S510: S— inverted-Abasic5′-cap, L-DNA 18/AS-AL. A 19-mer stabilized double strand RNA withinverted Abasic as 5′-cap, L-DNA at the 18 position of the sense strandand alternating 2′-OMe at positions 2, 4, 6, 8, 11, 13, 15, 17 & 19 onthe antisense strand.

Storage Conditions: −80° C.

siRNA formulated in PBS 33.3 mg/ml CASP2_(—)4_S510 in PBS (solution foreye drops)—

Description of the test material: Under sterile conditions, 300 mg dryCASP2_(—)4_S510 siRNA were dissolved in 15 ml of sterile doubledistilled water, to achieve a clear 20 mg/ml solution. The solution wasstored at −80° C. until use. The 20 mg/ml stock solution in doubledistilled water was then brought to a working concentration of 100 μg/3μg, in PBS, as follows:

1.7 mg CASP2_(—)4_S510 siRNA: 85 μl of 20 mg/ml stock of CASP2_(—)4 (1.7mg), were precipitated by 0.15M NaCl and EtOH, and dried under thetissue culture laminar (sterile conditions).

siRNA solution 33.3 mg/ml: 1.7 mg dry siRNA were dissolved in PBS ×1 toachieve 51 μl Quantity prepared: 1 tube of 1.7 mg in 51 μl of PBSStorage Conditions: freshly prepared

siRNA formulated in commercial lubricant solution Systane®: 33.3 mg/mlCASP2_(—)4_S510 in Systane (solution for eye drops)—Group II. Purchasedfrom Alcone Inc.; Batch #: Lot 165228F.

Description of the test material: Under sterile conditions, 300 mg ofCASP2_(—)4_S510 powder (Aligent, batch #Q02F08002N) were dissolved in 15ml of sterile double distilled water, to achieve a clear 20 mg/mlsolution. The solution was stored at −80° C. until use. The 20 mg/mlstock solution in double distilled water was then brought to a workingconcentration of 100 μg/3 μl, in formulation, as follows:

1.7 mg CASP2_(—)4_S510 siRNA: 85 μl of 20 mg/ml stock of CASP2_(—)4_S510(1.7 mg), was precipitated by 0.15M NaCl and EtOH, and dried under thetissue culture laminar (sterile conditions).

siRNA solution 33.3 mg/ml: 1.7 mg dry siRNA was dissolved in 51 μLcommercial lubricant solution (i.e. Systane®).

Quantity prepared: 1 vial of 1.7 mg siRNA in 51 μL Systane solution.

SiRNA formulated in glycerol+EDTA solution and in methyl cellulose (MC)Solutions: 33.3 mg/ml solution of CASP2_(—)4_S510 siRNA in Glycerol+EDTAformulation, and in 0.5%, 2% or 3% (w/v) methylcellulose & 1% (v/v)sterile glycerol & 0.01% (w/v) EDTA solution in pyrogen freewater—Groups III-VI

Description of the test material: Under sterile conditions, 300 mg ofCASP2_(—)4_S510 powder (Aligent, batch #Q02F08002N) were dissolved in 15ml of sterile double distilled water, to achieve a clear 20 mg/mlsolution. The solution was stored at −80° C. until use. The 20 mg/mlstock solution in double distilled water was then brought to a workingconcentration of 100 μg/3 μl, in formulation, as follows:

Seven vials of 1.7 mg CASP2_(—)4_S510 siRNA: 85 μl of 20 mg/ml stock ofCASP2_(—)4_S510 (1.7 mg), was precipitated by 0.15M NaCl and EtOH, anddried under the tissue culture laminar (sterile conditions).

Group III: 0.5% MC formulation: 1:7 mixture of solution A and solution B[12.5 μL of Solution A were mixed with 50 μL of Solution B and 37.5 pdWFI to obtain: 0.5% methylcellulose & 1% v/v sterile Glycerol & 0.01%w/v EDTA solution in pyrogen free water (final pH was approximately 7.4and osmolarity similar to human tear film)]

siRNA formulated in 0.5% MC formulation 33.3 mg/ml: 1.7 mg dry siRNAwere dissolved in 0.5% MC formulation that was prepared as described toachieve a 51 μL formulated solution of siRNA in 0.5% MC formulation.

Group IV: 1% MC formulation: 1:3 mixture of solution A and solution B[25 μL of Solution A were mixed with 50 μL of Solution B and 25 pd ofWFI to obtain: 1% methylcellulose & 1% v/v sterile Glycerol & 0.01% w/vEDTA solution in pyrogen free water (final pH was approximately 7.4 andosmolarity similar to human tear film)]

siRNA solution formulated in 1% MC formulation 33.3 mg/ml: 1.7 mg drysiRNA were dissolved in 1% MC formulation that was prepared as describedto achieve a 51 μL formulated solution of siRNA in 1% MC formulation.

Group V: 2% MC formulation: 1:1 mixture of solution A and solution B [5μL of Solution A were mixed with 50 μL of Solution B to obtain: 2%methylcellulose & 1% v/v sterile Glycerol & 0.01% w/v EDTA solution inpyrogen free water (final pH was approximately 7.4 and osmolaritysimilar to human tear film)]

siRNA solution formulated in 2% MC formulation 33.3 mg/ml: 1.7 mg drysiRNA were dissolved in 2% MC formulation that was prepared as describedto achieve a 51 μL formulated solution of siRNA in 2% MC formulation.

Group VI: 3% MC formulation: 1:1 mixture of solution A and solution C[50 μL of Solution A were mixed with 50 μL of Solution C to obtain: 3%methylcellulose & 1% v/v sterile Glycerol & 0.01% w/v EDTA solution inpyrogen free water (final pH was approximately 7.4 and osmolaritysimilar to human tear film)]

siRNA solution formulated in 3% MC formulation 33.3 mg/ml: 1.7 mg drysiRNA were dissolved in 3% MC formulation that was prepared as describedto achieve a 51 μL formulated solution of siRNA in 4% MC formulation.

Group VII: Glycerol+EDTA formulation: 50 μL WFI (Norbrook) were mixedwith 50 μL of Solution B to get: 1% v/v sterile Glycerol & 0.01% w/vEDTA solution in pyrogen free water.

siRNA solution formulated in Glycerol+EDTA formulation 33.3 mg/ml: 1.7mg dry siRNA were dissolved in Glycerol+EDTA formulation that wasprepared as described to achieve a 51 μL formulated solution of siRNA inGlycerol+EDTA formulation.

Quantity prepared: 7 vials, as follows:

1 vial of 1.7 mg siRNA in 51 μL PBS formulation

1 vial of 1.7 mg siRNA in 51 μL Systane® formulation

1 vial of 1.7 mg siRNA in 51 μL 0.5% MC formulation

1 vial of 1.7 mg siRNA in 51 μL 1% MC formulation

1 vial of 1.7 mg siRNA in 51 μL 2% MC formulation

1 vial of 1.7 mg siRNA in 51 μL 3% MC formulation

1 vial of 1.7 mg siRNA in 51 μL Glycerol+EDTA formulation

Storage Conditions: freshly prepared

Control Article(s)

1. PBS—Supplied by Biological industries (Manufacturer catalog#02-023-5A (For 10×PBS); Batch #619113).

2. Systane®—commercially available eye drop solution; Supplied by Alcon;Batch #: Lot 165228F; Quantity supplied: 15 ml; Storage Conditions: RT;Expiration Date: 02/2011.

Formulation Solution A—methylcellulose solution in pyrogen free waterSolution A) 0.4 g of methylcellulose 25 (ScienceLab.com, Cat# SLM2050)dissolved in final volume of 10 ml of hot boiled water (80-90° C.),(Norbrook), cooled down to room temperature.

Formulation Solution B—Sterile Glycerol & EDTA solution in pyrogen freewater Solution B) 332 μl of 60% glycerol (Sigma, Cat# G6279) & 2 μl EDTAsolution pH8 (prepared from Sigma, Cat# E9884) in 9.666 ml WFI(Norbrook).

Formulation Solution C—Concentrated methylcellulose solution in pyrogenfree water Solution C) 0.6 g of methylcellulose 25 (ScienceLab.com, Cat#SLM2050) dissolved in final volume of 10 ml of hot boiled water (80-90°C.), (Norbrook), cooled down to room temperature.

Stability Tests of siRNA Compounds in the Above Methyl CelluloseFormulations:

The stability of Casp2_(—)4 Q02F08002N siRNA was tested in Methylcellulose formulations according to the following protocol:

siRNA was diluted in the different formulations containing Methylcellulose, to a final concentration of 7 μM siRNA. The 0%,0.5%,1%, 2%and 3% MC formulations were incubated at Room Temperature. In addition,the 3% MC formulation was also incubated at 37° C. with and withoutnuclease inhibitor.

A 5-μL aliquot of each solution was transferred to 15 μL of10×TBE-loading buffer after incubation at the following time points:0.10′, 0.5, 1, 1.5, 3, 6 h. The solution was then frozen in liquidnitrogen, and stored at −20° C.

4 μL of each sample was loaded onto a non-denaturing 20% polyacrylamidegel and electrophoresis was performed at 80V for 2.5 h.

For siRNA visualization the gel was stained with Ethidium bromidesolution (1.0 μg/μL).

As a positive control for gel migration of a non-degraded siRNA, 5 μL ofa 7 μM tested siRNA solution in PBS was transferred to 15 μL of10×TBE-loading buffer and loaded onto the gel. Then, the sample wasfrozen in liquid nitrogen and stored at −20° C.

As a reference to the migration pattern of a degraded single strand (ss)siRNA, a non-relevant single strand siRNA was prepared.

Results: The Casp 2_(—)4 siRNA compound was stable in all formulations.

The In Vivo Study:

Test System:

Animals used: Rats; Strain: Adult, Sprague-Dawley; Modification: SD

Source: Harlan, Jerusalem Israel

Age: 8-10 weeks; Body Weight Range: 220-270 gr

Sex: Male; Group Size: n=6; Total number of animals: 54

Animal Husbandry: Diet: Animals were provided an ad libitum commercialrodent diet and free access to drinking water.

Environment:

(i) Acclimatization of at least 5 days.

(ii) All the animals were confined in a limited access facility withenvironmentally-controlled housing conditions throughout the entirestudy period, and maintained in accordance with approved standardoperating procedures (SOPs). Animals were provided ad libitum acommercial rodent diet (Harlan Teklad 2018S Global 18% Protein RodentDiet) and filtered, chlorinated and acidified water.

Rats were kept in microisolator cages with filter top, 1-6 Rats/cage.The cages were maintained under controlled environmental conditions oftemperature (20-24° C.), relative humidity (30-70%), a 12-hr light/12-hrdark cycle, monitored by the control computer, throughout the studyperiod.

Experimental Design

Experimental setup included 9 experimental groups (6 rats per group). EDwere applied bilaterally. Different formulations were used (Study DesignTable C11). Termination time point (3 hrs) post siRNA administration:Termination setup was performed according to the study design Table C11.Retinal concentration of CASP2_(—)4 siRNA was determined by qPCR.

TABLE C11 Study Design Bilateral Delivery siRNA Application Term. TimeGroup Group Route Formulation Compound (dose/volume/eye) Point (hrs)Size I ED PBS Casp2_4_S510 100 μg/3 μl 3 6 II ED Systane Casp2_4_S510100 μg/3 μl 3 6 III ED MC0.5% Casp2_4_S510 100 μg/3 μl 3 6 IV ED MC1%Casp2_4_S510 100 μg/3 μl 3 6 V ED MC2% Casp2_4_S510 100 μg/3 μl 3 6 VIED MC3% Casp2_4_S510 100 μg/3 μl 3 6 VII ED Glycerol + Casp2_4_S510 100μg/3 μl 3 6 EDTA * VIII ED PBS — — 3 6 IX Intact — — — — 6 * Glycerol +EDTA formulation (MC free formulation) = 1% v/v sterile glycerol & 0.01%w/v EDTA solution in pyrogen free water

Anesthesia: In the course of the experiment, All ED treated animals wereanesthetized with Equithesine (I.P. 4 ml/kg).

Eye Drop Delivery A 3 μl sample volume of the test article or vehiclewere applied to the corneal surface bilaterally to the anesthetizedanimals, by a blunt pipette tip (filter tips 10 μl sterile (short)). Theanimals were placed in a warm environment to prevent anesthesia-inducedhypothermia. Termination of animals from all study groups was accordingto study design (time point 3 hrs). Tissues were collected according tostudy design.

Scheduled euthanasia: All animals were deeply anaesthetized andeuthanized according to the study design (Table C11, Termination).

Perfusion setup: Rats were perfused transcardially with PBS (20-50rpm/min following 2-3 min).

Tissue Collection: After perfusion, both eyes (left and right) werewashed with PBS, were enucleated and stored on ice. The eyes weredissected using a binocular microscope, and optionally gross pathologywas graded according to the sample grading scale. The cornea wasdissected by a cut along the limbus, lens was gently removed, and theretina and vitreous were carefully separated from the sclera. Wholeretinas and vitreous bodies (humor) were collected into two separateappropriate and properly marked test tubes. Dissected retinas werewashed in a large volume of PBS (each retina in a separate tube withfresh PBS), extra liquid was removed with Kimwipes and retinas weresnap-frozen in liquid nitrogen. Retina and Vitreous body samples weresubjected to RNA extraction.

Evaluation:

RNA Extraction: Retina: RNA was extracted from each retina sampleindividually (left and right) by double extraction. The RNA wastransferred for cDNA preparation and qPCR analyses.

Vitreous: Material for Casp2_(—)4_S510 siRNA detection from vitreous wasobtained using the following protocol: Each rat vitreous was around 10μl. 500 μl of reagent A EZ-RNA II (Biological Industries Cat no.20-410-100) were added to each vitreous. The sample was homogenized, and10 μg tRNA (1 μl from 10 mg/ml stock) were added. The sample was storedfor 5 minutes at room temperature. Subsequently EZ-RNA II B: 400 pd andEZ-RNA II C: 90 μl were added and mixed well. The sample thus obtainedwas stored for 10 min at room temperature then centrifuged 12000 g for15 min at 4° C.

The upper phase was transferred to a fresh tube to which isopropanol:500 μl and Linear Acrylamide: 5 μl were added. The sample was storedovernight at −20° C., centrifuged at 12000 g for 20 min at 4° C., washedtwice with 75% ethanol. The pellet was dissolved in 15 μl DDW.

siRNA Quantification: The quantity of the siRNA in retinas and vitreoushumor (siRNA quantification was performed per vitreous) was examined byqPCR siRNA according to Quark's standard operating procedures.

Results

The study showed that the following quantities of siRNA were detected inthe rat retina three hours post application of siRNA non-invasivelyadministered in vivo with the different formulations:

TABLE C12 CASP2 siRNA Normalized (miRNA) quantity in fmle/1 μg RNA GroupAbbreviation N Mean Std IX I-N/A 6 0.005 0.005 VIII P-PBS 6 0.056 0.124VII C-MC(0) 6 6.303 3.921 XA C-MC(1/2) 4 17.831 16.628 XIA C-MC(1) 65.677 4.235 V C-MC(2) 6 6.963 8.042 VI C-MC(3) 5 7.653 6.674 IIC-Systane 5 5.109 2.492 I C-PBS 6 4.228 6.758

MC(0)-MC(3)—formulations including increasing concentrations from 0 to3% of Methyl Cellulose.

Conclusion: All groups that were treated by CASP2 siRNA show positivequantities of the siRNA (>4 fmole) in target retinal tissue. Nosignificant difference (P-value=0.8425) were found between theformulation groups.

Non-Invasively Administered siRNA Induces In-Vivo Knockdown (KD) ofTarget Gene Associated with Apoptosis in Target Ocular Tissue

Example 8 Determination of In-Vivo Knockdown Activity of siRNA Againstp53 Prepared for Non-Invasive Administration by Eye Drops in Rat Retina(siRNA Compound Formulated in MC 2% (Formulation “A”) Objective

The objective of the study was to determine knockdown activity ofnon-invasively delivered siRNA compound targeting p53 mRNA in the ratneural retina. The siRNA compound was formulated for non-invasiveadministration by eye drops. Knockdown activity was assessed by proteinlevel determination using ELISA method.

Test Article

a. Substance (unformulated siRNA compound): QM5 (siRNA Against Mouse/Ratp53)

b. Formulated siRNA compound for non-invasive (eye drop) delivery(groups 2 & 5 in Table C13): 100 μg/3 μl solution of QM5 siRNA in 2%(w/v) methylcellulose & 1% (v/v) sterile glycerol & 0.01% (w/v) EDTAsolution in pyrogen free water.

c. Formulated siRNA compound for intravitreal injection (groups 1 & 4 inTable C13): 280 μg of QM5 siRNA in 140 μl of PBS

d. Formulated vehicle solution for non-invasive (eye drop) delivery(groups 3 & 6 in Table C13): 2% methylcellulose & 1% v/v sterileglycerol & 0.01% w/v EDTA solution in pyrogen free water.

Test System:

Adult Male, Sprague-Dawley (SD) Rats Harlan, Jerusalem Israel, 6-8 weeksold, 160-180 gr each.

Experimental Design

Study design: Unilateral axotomy (left eye, OS) was performed in eachanimal from groups 1-6.

Groups 2, 3, 5& 6: Test compound (100 μg test article in 3 μl offormulated vehicle—groups 2&5) or formulated vehicle alone (groups 3&6)were applied as eye drops every day starting on day 0 (immediately afteraxotomy). Experimental groups were terminated according to Table C13.Left and Right eye samples from group 7 served as an intact normalcontrols.

Groups 1 & 4: Three minutes following axotomy (at day 0), 20 μg of siRNAcompound in 10 pd of PBS vehicle were applied by microinjection into thevitreous. The microinjection into the vitreous was performedperpendicular to the sclera, using an Insulin micro-injector (0.3 ml).Animals were terminated according to Table C13.

TABLE C13 Study Design: Intravitreal Axotomy injection ED (OS) Termi-Group (unilat. (OS) (dose/ (dose/volume/ nation Group Size OS)volume/eye) eye) (days) 1 6 Yes QM5 20 N/A 1 μg/10 μl 2 6 Yes N/A QM5100 μg/3 μl 1 3 4 Yes N/A Formulated 1 vehicle 4 6 Yes QM5 20 N/A 3μg/10 μl 5 6 Yes N/A 3x QM5 100 μg/3 3 μl 6 4 Yes N/A 3x Formulated 3vehicle 7 4 N/A N/A N/A N/A

(see Tables C14-C17 hereinbelow and FIGS. 11A and 11B)

p53 Signal According to Kidney W.C.E Standard Curve

TABLE C14 p53 signals values according to Kidney W.C.E standard curveP53 Signal retina Operation Treatment 1 days 3 days Axotomy QM5Intravitreal 1.00 1.84 Axotomy QM5 Eye Drops 0.75 1.41 Axotomy VehicleEye Drops 1.93 Missing None Non Treated 2.01 2.01

TABLE C15 % p53 signals ratio from non-treated animals % signal ratiofrom non-treated retina Axotomy QM5 Intravitreal 50% 92% Axotomy QM5 EyeDrops 37% 70% Axotomy Vehicle Eye Drops 96%  0%

p53 Signal According to GST-Hp53

TABLE C16 p53 signal values according to GST-p53 standard curve P53Signal retina Operation Treatment 1 day 3 days Axotomy QM5 Intravitreal0.0159 0.0214 Axotomy QM5 Eye Drops 0.0095 0.0149 Axotomy Vehicle EyeDrops 0.0227 Missing None Non Treated 0.0233 0.0233

TABLE C17 % P53 signals ratio from non-treated animals % Signal ratiofrom non-treated retina Axotomy QM5 Intravitreal 68% 92% Axotomy QM5 EyeDrops 41% 64% Axotomy Vehicle Eye Drops 97% Missing

Conclusion: These studies showed that non-invasively delivered siRNAcompound targeting p53 mRNA in the rat neural retina induces knock downof the p53 protein.

siRNA Compounds Induce Ocular Neuroprotection In-Vivo in Animal Modelsof Ocular Neuronal Injury

Example 9 Evaluation of Ocular Neuroprotective Induction by siRNACompound Targeting Caspase 2 in the ONC Model after IVT Injection

Study objectives: To evaluate ocular neuroprotective efficacy of siRNAtargeting Caspase 2 in the ONC model following intravitreal injection(s)

Methods Retrograde Labelling of RGCs.

The Fluorogold tracer is transported retrogradely (from brain to eye)along RGC axons resulting in complete and specific labeling of all RGCs.For the purpose of this study, RGCs were labeled by application of theretrograde tracer FluoroGold (2%, Fluorochrome, Englewood, Colo.) in thesuperior colliculus. Briefly, a window was drilled in the scalp abovethe coordinates 6 mm rostral to the bregma and 1.2 mm lateral to themidline in both hemispheres. Using a Hamilton syringe, 3 pd of theFluoroGold was injected into superior colliculus 3.8 mm, 4 mm, and 4.2mm below the bony surface at a rate 1 μl/min at each of the threedepths. The needle was then slowly withdrawn and the skin was sutured.In adult rats, the time required to obtain full labeling of all RGCsfollowing this procedure was ˜1 week. For this reason, ONC was performedone week after retrograde labeling of RGCs.

Optic nerve crush. The orbital optic nerve (ON) of anaesthetized adultsWistar rats was exposed through a supraorbital approach, the meningessevered and all axons in the optic nerve (ON) transected by crushingwith calibrated forceps for 10 seconds, 2 mm from the lamina cribrosa.

Intravitreal (WT) injection. One or two 20 μg doses (in 10 pd of PBS) oftest or control siRNAs or 10 μl of PBS vehicle were microinjected intothe vitreous body 2 mm anterior to the nerve head, perpendicular to thesclera, using a glass micropipette. IVT administration is shown as acontrol and or for initial validation of a target gene.

Quantification of RGC survival. At termination, experimental animalswere perfused transcardially with 4% paraformaldehyde. The eyes with theoptic nerve were enucleated, the cornea was dissected with the blade andlens/vitreous were gently removed. Both retinas were dissected out,fixed for an additional 30 min and flat-mounted vitreal side up on aglass slide for examination of the ganglion cell layer. RGCs wereexamined under the fluorescence microscope with an UV filter (365/420nm). The number of retrogradely fluorescent RGCs were determined by twodifferent, independent and “blinded” investigators counting them in 16distinct areas (four areas per retinal quadrant at three differenteccentricities of one-sixth, one-half, and five-sixths of the retinalradius). Microglia that may have incorporated FluoroGold afterphagocytosis of dying RGCs was distinguished by their characteristicmorphology and excluded from quantitative analyses.

Part 1

Test article: CASP2_(—)4 siRNA—a double-stranded 19-mer oligonucleotidechemically modified by 2′O-methylation on both strands. Other compoundsincluded siRNA chemically modified with L-DNA at the 3′ terminus of thesense strand or 2′5′ bridges at the 3′ terminus. Targets the caspase2gene in multiple species.

Control article: PBS

Design: Retinal ganglion cells (RGC) were selectively labeled first byapplication of the retrograde tracer FluoroGold to the superiorcolliculus. One week later, animals were subjected to optic nerve crushinjury (ONC). The quantifications of surviving RGCs were carried out at7 and 30 days after ONC by counting FluoroGold-labelled RGCs onflat-mounted retinas. Table C18 shows the groups treatments.

TABLE C18 Group Treatments Groups Right eye (n = 4) Left eye (n = 4)Termination 1 and 2 20 μg siCasp2_4 20 μl PBS Day 7 on day 0 on day 0 3and 4 20 μg siCasp2_4 20 μl PBS Day 30 on days 0 and 10 on days 0 and 10

Results: The mean number of surviving RGCs in eyes treated with 20 μgCasp2_(—)4 siRNA and subjected to optic nerve crush was 2040±35cells/mm2 at 7 days and 298±25 cells/mm2 at 30 days post injury. Thesecounts were significantly greater than the mean RGC counts in eyestreated with PBS and subjected to nerve crush, which was 941±27cells/mm2 at 7 days and 41±7 cells/mm2 at 30 days. In the nonsurgicalcontrol eyes, the mean number of RGCs was 2253±104 cells/mm2, which iscomparable to the average number of RGCs reported in literature. TableC19 provides the results of this study.

TABLE C19 Mean numbers of survived RGC at the different time points postinjury Time post ONC 7 days 30 days Treatment PBS Casp2_4 PBS Casp2_4Mean survival 941.347 2039.672 41.040 298.051 RGC (cells/mm2) SD 27.03834.766 7.219 25.401 % of RGC survival 26.659 57.764 1.162 8.441 fromtotal RGC SD 0.766 0.985 0.204 0.719

All data are given as mean±standard deviation. Values were comparedusing one-way analysis of variance (ANOVA) and considered assignificantly different at P<0.02.

Conclusions: The increased survival of the RGCs was not due to theneuroprotective effects of anesthesia (ketamine is an antagonist of theN-methyl-D-aspartate receptor, and xylazine is an α2-adrenergic receptoragonist), since identically anesthetized animals in the control grouphad significantly lower RGC counts than did animals in the Casp2_(—)4siRNA treated group, but due to neuroprotective effect of silencingpro-apoptotic Caspase 2 activation and up regulation following ONCinjury, by treatment with siRNA compound targeting the Caspase 2 gene.

Part 2

Test article: CASP2_(—)4L siRNA—a double-stranded 19-mer oligonucleotidechemically modified by 2′O-methylation on the antisense strand and L-DNAon the sense strand. Targets the caspase 2 gene of multiple species.

Control articles:

-   -   PBS    -   siRNA targeting GFP—a double-stranded 21-mer oligonucleotide        stabilized by 2′O-methylation on both strands.    -   CNL—siRNA with no match to any known mammalian transcript; a        double-stranded 19-mer oligonucleotide chemically modified by        2′O-methylation on both strands.

Design. Retinal ganglion cells (RGC) were selectively labeled first byapplication of the retrograde tracer FluoroGold to the superiorcolliculus. One week later, animals were subjected to optic nerve crushinjury (ONC). The quantifications of surviving RGCs were carried out atday 7 after ONC by counting FluoroGold-labelled RGCs on flat-mountedretinas. Test or control articles were injected at the time of ONC.Similar experiments were performed in order to test activity andefficacy of siRNA administered via eye drops (see next example). TableC20 shows groups treatments.

TABLE C20 Groups Treatments Groups Right eye (n = 4) Left eye (n = 4)Termination 1 and 2 20 μg siCasp2_4 20 μl PBS Day 7 on day 0 on day 0 3and 4 20 μg siGFP 20 μl siCNL_1 Day 7 on day 0 on day 0

Results: The mean number of surviving RGCs in eyes treated with 20 μgCasp2_(—)4 siRNA and subjected to optic nerve crush was 2085±40cells/mm2 at 7 days post injury. These counts were significantly greaterthan the mean RGC counts in eyes treated with either PBS or GFP siRNA orCNL_(—)1 siRNA and subjected to nerve crush, which was 901±50 cells/mm2,922±38 cells/mm2, 898±42 cells/mm2 respectively, at 7 days. In thenonsurgical control eyes, the mean number of RGCs was 2196±110cells/mm2, which is comparable to the average number of RGCs reported inliterature. Table C21 shows results. All data are given as mean±standarddeviation. Values were compared using one-way analysis of variance(ANOVA) and considered as significantly different at P<0.01.

TABLE C21 Mean numbers of survival RGC 7 days post injury (n = 4retinas/group). Treatment PBS siGFP siCNL_1 siCasp2 Mean survival RGC901.3338 922.3666 898.4268 2084.815 (cells/mm2) SD 49.74134 38.0405942.12429 40.03638 % of RGC survival 25.52577 26.12142 25.44345 59.04197from total RGC SD 1.408675 1.07731 1.19296 1.13383

Conclusions: The increased survival of the RGCs was due toneuroprotective effect of silencing pro-apoptotic Caspase 2 activationand up-regulation following ONC injury, by treatment with siRNAtargeting Caspase 2 gene. The Casp2_(—)4 siRNA molecules havingdifferent structural modifications/motifs show similar neuroprotectiveeffect on RGC survival range.

Example 10 Evaluation of Ocular Neuroprotective Efficacy of siRNACompound Targeting Caspase 2 in the IOP Model

The objective of the current study was to establish the neuroprotectiveeffect of a single injection of 20 μg of CASP2_(—)4 compound or negativecontrol GFP siRNAs in the pre-clinical rat glaucoma IOP model.

Study Outline

In animals from all study groups, RGCs were retrograde labeled using thefluorescent tracer DiI(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine) applied to bothsuperior colliculi. A week later, IOP was unilaterally increased in theleft eyes of animals from groups 2 to 5. IOP was monitored every otherday in the operated (left) and contralateral (right) eyes during 2weeks. Two weeks following hypertonic saline injection, vehicle (PBS) orsiRNA (20 μg in PBS, either siRNA targeting Casp2 or a negative controlsiRNA targeting the non-mammalian gene, Green Fluorescent Protein), wasadministered by intravitreal (IVT) injection to the left (operated)eyes. The contralateral non-operated eyes received no IVT injection. Inaddition, two weeks after hypertonic saline injection, the animals fromgroup 2 were sacrificed and DiI-labeled RGCs were counted in flat mountsof left retinas under fluorescence microscopy, in order to assess theextent of RGC loss at the time of siRNA injection. For all groupsreceiving WT injection, IOP was measured once during the 3rd week of theexperiment to confirm its increased state in the operated eyes. At 3weeks of increased IOP, or at 1 week after the test and control articleinjections, the animals were sacrificed and DiI-labeled RGCs werecounted in flat mounts of left retinas under fluorescence microscopy.Right retinas served as internal controls for labeling quality. Aseparate group of naïve rats in which RGC's were retrogradely labeled atthe experiment commence was used as an intact control to provide areference for normal RGC densities. 4 rats from this group weresacrificed at 2 weeks of ocular hypertension (together with group 2) andthe remaining 9 rats—at 3 weeks (together with groups 3 to 5). Theexperimental design is shown in Table C22 below.

Animals

Species: Rats Strain: Brown Norway Rats Modification: N/A Source:Charles River (Canada) Age: Adult, retired breeders, 10-12 months of ageBody Weight Range: 300-400 g Sex: Males Animal Husbandry: Diet:Environment: All animal procedures were performed in accordance with theguidelines of the Canadian Council on Animal Care for the use ofexperimental animals (http://www.ccac.ca/). Environment: (i)Acclimatization of at least 5 days. (ii) All the animals were confinedin a limited- access facility with environmentally con- trolled housingconditions throughout the entire study period, and maintained in accor-dance with University of Montreal approved standard operating procedures(SOPs).

Materials and Equipment

Substance (unformulated compound) CASP2_(—)4_S510 (CASP2_(—)4; CASP2SIRNA; siRNA Against Caspase2 mRNA)

-   -   Supplied by Agilent    -   Description of the test material: A 19-mer chemically modified        blunt-ended duplex having two separate strands, with a sense        strand (SEN) comprising unmodified ribonucleotides (upper case        letters), an L-deoxyribonucleotide at position 18 (bold,        underlined) and inverted deoxyabasic moiety (iB) present at the        5′ terminus of the SEN strand; and with an antisense strand (AS)        comprising unmodified ribonucleotides (upper case letters), and        2′OMe sugar modified ribonucleotides (lower case letters) at        positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 as shown in Formula        I.    -   Quantity supplied: 300 mg    -   Storage Conditions: −80° C.

Control Substance (unformulated compound) GFP_(—)5_S763 (siRNA AgainstGFP mRNA)

-   -   Outsourcing (manufacturer's name): Agilent    -   Manufacturer's catalog #N/A    -   Quantity supplied: 220.8150 mg    -   Storage Conditions: −80° C.; Expiration Date: ND

Test/Control Article (formulated) CASP2_(—)4/GFP_(—)5 20 μg/5 μL of PBS

0.5 mg was diluted into 125 μl to achieve a stock solution of 4 μg/μl.These were subsequently aliquoted to 5 μl per tube and stored at −80° C.

Vehicle—PBS.

Multicell Phosphate buffered saline, solution 1×, without calcium,without magnesium (CAT. No: 311-010-EL).

Fluorescence microscopy was performed using a Zeiss Axioskop 2 Plusmicroscope (Carl Zeiss Canada, Kirkland, QC), images were captured witha CCD camera (Retiga, Qimaging) and processed with Northern Eclipseimage analysis software (Empix Imaging, Mississauga, ON).Microphotographs were taken at 25× magnification.

Experimental Procedure

Surgery was be performed in adult male Brown Norway rats, retiredbreeders, between 10-12 months of age (300-400 g), under generalanesthesia by intraperitoneal injection of 1 mL/Kg standard rat cocktailconsisting of Ketamine (100 mg/mL), xylazine (20 mg/mL) and acepromazine(10 mg/mL).

Ocular hypertension surgery: Unilateral and chronic elevation of IOP wasinduced by using the Morrison model, involving an injection ofhypertonic saline into a episcleral vein, leading to a blockade of theaqueous humor outflow pathways. This procedure lead to gradual increaseof eye pressure and progressive death of RGS. All the animals in thisstudy received only a single saline vein injection. The eye selected forthe procedure was adapted with a plastic ring applied to the ocularequator to confine the saline injection to the limbal plexus. A microneedle (30-50 μm in diameter) was used to inject 50 μL of sterile 1.85 MNaCl solution through one episcleral vein. The plastic ring temporarilyblocked off other episcleral veins forcing the saline solution into theSchlemm's canal to create isolated scarring. Animals were kept in a roomwith constant low fluorescent light (40-100 lux) to stabilize circadianIOP variation (8).

Measurement of intraocular pressure (IOP): IOP from glaucomatous andnormal (contralateral) eyes was measured in awake animals using acalibrated tonometer (TonoPen XL, Medotronic Solan, Jacksonville, Fla.).IOP was measured every other day for the first two weeks after ocularhypertension surgery; and at least once between week 2 and week 3 afterocular hypertension surgery. Eyes become fragile after intraocularinjections in conditions of high IOP, for this reason the number of IOPmeasurements was limited as this involves putting additional pressure onthe cornea to get a reliable reading. The mean IOP (mmHg±S.E.M) per eyewas considered as the average of all IOP readings since the onset ofpressure elevation. The maximum IOP measurements in each individual eye,glaucomatous or normal contralateral eye was defined as the peak IOP andthis value was used to estimate the mean peak IOP for each group

Calculation of intraocular pressure (IOP): The mean IOP (mmHg±S.E.M) pereye was considered as the average of all IOP readings since the onset ofpressure elevation. The maximum IOP measurements in each individual eye,glaucomatous or normal contra lateral eye was defined as the peak IOPand this value was used to estimate the mean peak IOP for each group.The positive integral IOP was calculated as the area under the IOP curvein the glaucomatous eye minus that of the fellow normal eye from ocularhypertension surgery to euthanasia. Integral IOP represents the total,cumulative IOP exposure throughout the entire experiment.

Study Design:

One week prior to induction of glaucoma, RGCs were retrogradely labeledusing the fluorescent tracer DiI applied to both superior colliculi.

One week later, unilateral elevation of IOP was induced by injection ofa hypertonic saline solution into an episcleral vein. This procedure wasreferred to as ocular hypertension surgery.

In a first experiment, prior to efficacy studies, the status of RGC lossin “no injection” groups at 2 and 3 weeks after ocular hypertensionsurgery (4 rats/group, total=8 rats) was assessed.

Exactly 2 weeks after ocular hypertension surgery, a single intravitrealinjection of each siRNA was performed.

Animals were euthanized and retinas prepared for analysis of RGCsurvival at exactly 3 weeks after ocular hypertension surgery.

The density of surviving RGCs was quantified in 12 standard retinalareas.

TABLE C22 Study Design In- IVT Injection (μL/ Termination creased eye) 2weeks post Dose (Weeks Post Group n IOP IOP induction Volume IOP) 1 13No No N/A N/A (Intact) (4 + 9) 2 4 Yes No N/A 2 3 6 Yes PBS Vehicle 5 μL3 4 6 Yes Negative Control 5 μL 3 siRNA, 20 μg 5 6 Yes CASP2 SIRNA, 20 5μL 3 μg *NA—non-applicable, Group size: n = 5

Quantification of surviving RGC soma: Quantification of RGC bodies wasperformed in duplicate and in a masked fashion. For RGC density counts,rats were deeply anesthetized and then perfused transcardially with 4%paraformaldehyde (PFA) in 0.1 M phosphate buffer and both eyes wereimmediately enucleated. Retinas were dissected and flat-mounted on aglass slide with the ganglion cell layer side up. Under fluorescencemicroscopy, DiI-labeled neurons were counted in 12 standard retinalareas as described.

Results & Discussion

TABLE C23 Percent RGC from intact inference table Treat N Mean Std CVDunnett P-value PBS 6 62.37 5.86 9.40 CASP2_4 6 81.71 14.28 17.48 0.052wIOP 4 71.25 6.75 9.47 0.19 GFPsiRNA 6 65.75 4.97 7.57 0.68 intact 13100.00 2.84 2.84 0.00

Comparisons to PBS Treated Group

Preservation of RGC in CASP2_(—)4 siRNA-treated eyes with increased IOPwas significantly better than in the PBS treated group (1.31 fold higherfrom the PBS result; p-value=0.05). In GFP siRNA treated group nosignificant difference was found (p-value=0.68) with respect to PBStreated group.

No significant difference between IOP (2 weeks) to IOP (3 weeks) treatedby PBS (p-value=0.19).

Comparisons to IOP (2 Weeks) Group

The intact group has significantly higher RGC density per mm2 than IOP(2w) group (p-value=0.00). No significant differences in RGC density permm2 were found between IOP (2w) group and other IOP groups that weresacrificed after 3 weeks (p-value range from 0.16 to 0.44).

Comparisons to Intact Group

The intact group has significantly higher RGC density per mm2 than IOPgroups that were treated by PBS, GFP siRNA and IOP(2w) without treatment(p-value<0.01). No significant differences in RGC density per mm2 werefound between IOP group treated by CASP2_(—)4 siRNA compared to intactgroup (p-value=0.06).

These data demonstrate that a siRNA targeting Caspase2 providesprotection of RGCs from damage induced by increased intraocular pressureand its administration at 2 weeks of increased IOP leads to cessation offurther RGC loss.

IOP measurements were carried out as described above.

IOP: The Dunnett approach was used for comparison of the results to PBStreated group. Additional parameters were calculated and analyzed suchas: AUC (area under the curve), Mean IOP and Max(IOP) along the durationof measurements.

Most animals show significant increase in IOP at 1-2 weeks after ocularhypertension surgery.

No significant differences in IOP levels were found among operated eyesin treated groups (0.7746). Likewise, no differences in IOP levels werefound among contra lateral eyes in treated groups and intact eyes.

The results show that Casp2 siRNA induced neuroprotection even as IOPremained elevated in all treatment groups.

Results: No significant differences between groups were found in allthree calculated IOP parameters

Example 11 Evaluation of Ocular Neuroprotective Efficacy of siRNACompound Targeting Caspase 2 in the Axotomy Model

The purpose of the present efficacy studies was to use a model of RGCapoptosis induced by axotomy of the optic nerve (ON) in adultSprague-Dawley rats. The onset and kinetics of RGC death in this modelsystem are very reproducible and allow for the establishment of theneuroprotective efficacy of Casp2_(—)4 siRNA in vivo. Using this method,the time course of RGC death follows a predictable course: cell deathbegins on day 5 and proceeds to the rapid loss of more than 90% of theseneurons by 2 weeks.

Methods

Retrograde labeling of RGCs: For the purpose of this study, RGCs werelabeled by application of the retrograde tracer FluoroGold (2%,Fluorochrome, Englewood, Colo.) in the superior colliculus. Briefly,both superior colliculi were exposed and a small piece of gelfoam soakedin FluoroGold was applied to their surface. In adult rats, the timerequired to obtain full labeling of all RGCs following this procedure is˜1 week. For this reason, optic nerve axotomy and intraocular injectionof siRNA molecules were performed one week after retrograde labeling ofRGCs.

Optic nerve axotomy: The entire population of RGCs were axotomized bytransecting the optic nerve close to the eye (0.5 to 1 mm). Retinalfundus examination was routinely performed after each axotomy to checkthe integrity of the retinal circulation after surgery. Animals showingsigns of compromised blood supply were excluded from the study.

For intravitreal injection 10 μg in 5 pd of PBS each of the reagents,either Casp2_(—)4 siRNA or GFP siRNA were microinjected into thevitreous body 2 mm anterior to the nerve head, perpendicular to thesclera, using a glass micropipette at the time of surgery, day 0, andthen repeated at day 7.

Quantification of RGC survival: Experimental and control animals wereperfused transcardially with 4% paraformaldehyde at 14 days after opticnerve axotomy. The left retinas (treated) and the right retinas(untreated controls) were dissected out, fixed for an additional 30 minand flat-mounted vitreal side up on a glass slide for examination of theganglion cell layer. RGCs backfilled with FluoroGold were counted in 12standard retinal areas. Microglia that may have incorporated FluoroGoldafter phagocytosis of dying RGCs were distinguished by theircharacteristic morphology and excluded from our quantitative analyses.

Experimental Design

Test article: CASP2_(—)4L siRNA: a double-stranded 19-meroligonucleotide chemically modified by 2′O-methylation of sugar residueson the antisense strand and L-DNA on the sense strand. Targets caspase2gene in multiple species.

Control Articles

PBS

siRNA targeting GFP—a double-stranded 21-mer oligonucleotide chemicallymodified by 2′O-methylation on both strands.

CNL—siRNA with no match to any known mammalian transcript; adouble-stranded 19-mer oligonucleotide chemically modified by2′O-methylation on both strands. Table C24 shows groups treatments.

TABLE C24 Groups Treatments Time of ad- Time of No. of ministrationanalysis animals Sample SiRNA (post (post per prepa- SiRNA Dose axotomy)axotomy) group ration SiGFP, 10 ug Time 0 2 weeks 8 Flat mount axotomy(OS) X 2 1 week SiCasp2_4, 10 ug Time 0 2 weeks 8 Flat mount Axotomy(OS) X 2 1 week

Results: The mean number of surviving RGCs in eyes treated with twointravitreal injections of 10 μg Casp2_(—)4 siRNA and subjected toaxotomy was 533±24 cells/mm2 at 14 days post injury. These counts weresignificantly greater than the mean RGC counts in eyes treated with GFPsiRNA at the similar regiment and subjected to axotomy, which was 130±7cells/mm2 at 14 days. In the nonsurgical control eyes, the mean numberof RGCs was 2138±91 cells/mm2, which is comparable to the average numberof RGCs reported in literature.

TABLE C25 Mean numbers of survival RGC 14 days post injury (n = 6retinas/group) Treatment GFP Casp2_4 Mean survival RGC (cells/mm2) 130533 SE 7 24 % of RGC survival from total RGC 6 25 SE 0.32 1.12

Data analyses and statistics were performed using the GraphPad Instatsoftware by one-way analysis of variance (ANOVA). All data are given asmean±SE, significantly different at P<0.02.

Conclusions: The increased survival of the RGCs was due toneuroprotective effect by silencing pro-apoptotic Caspase 2 activationand up regulation following axotomy by treatment with siRNA targetingCaspase 2 gene.

Discussion: In the present studies, the Casp2_(—)4 siRNA wasneuroprotective for at least 30 days in an optic nerve crush model andfor 14 days in axotomy model of RGC loss. Optic nerve crush and axotomyexperiments provide a realistic model of acute optic neuropathies.

Non-Invasive Administration of siRNA Compounds Induces OcularNeuroprotection In-Vivo in Animal Models of Ocular Neuronal Injury

Example 12 Evaluation of Ocular Neuroprotective Efficacy ofNon-Invasively Administered siRNA Compound Targeting Caspase 2 in theONC Model

Test article: CASP2_(—)4 siRNA (compound of Formula I)

Control Articles:

-   -   Methyl Cellulose    -   CNL—siRNA with no match to any known mammalian transcript; a        double-stranded 19-mer oligonucleotide stabilized by        2′O-methylation on both strands.

Design: Retinal ganglion cells (RGC) were selectively labeled first byapplication of the retrograde tracer FluoroGold to the superiorcolliculus. One week later, animals were subjected to optic nerve crushinjury (ONC). Eye drops were applied every other day during one week (3times over all). 100 μg/3 μl CNL_(—)1, or Casp2_(—)4 siRNA or 3 μl of MCvehicle were applied, the first dose was applied 10 minutes after ONC.The quantifications of surviving RGCs were carried out at day 7 afterONC by counting FluoroGold-labelled RGCs on flat-mounted retinas.

Results: The mean number of surviving RGCs in eyes treated with 20 μgCasp2_(—)4 siRNA and subjected to optic nerve crush was 445±17 cells/mm2at 7 days post injury. These counts were significantly greater than themean RGC counts in eyes treated with either PBS or CNL_(—)1 siRNA andsubjected to nerve crush, which were 337±11 cells/mm², 341.6±13cells/mm², respectively, at 7 days. Table C26 shows results. All dataare given as mean±standard deviation. Values were compared using one-wayanalysis of variance (ANOVA) and considered as significantly differentat P<0.01.

TABLE C26 Mean numbers of survival RGCs 7 days post injury. CNL_1Casp2_4 MC vehicle Average 341.625 145.4375 337.5 SD 13.03265 17.22233111.3389342 cells/mm2 934.6785 1218.7073 923.392613 Sd 35.65705 17.11992131.0230758 RGC/retina 26470.1 34513.789 26150.4788 SD 1009.808 1334.4362878.573505 % total 26.4701 34.513789 26.1504788 SD 1.009808 1.33443620.87857351 Average 341.625 145.4375 337.5 SD 13.03265 17.22233111.3389342

Conclusions: The increased survival of the RGCs was due toneuroprotective effect of silencing pro-apoptotic Caspase 2 followingONC injury by treatment with eye drops containing siRNA targetingCaspase 2 gene in a methyl cellulose formulation.

Example 13 Evaluation of Ocular Neuroprotective Efficacy ofNon-Invasively Administered siRNA Compound Targeting Caspase 2 in theIOP Model Experimental Setup:

Experimental animals: All animal procedures are performed in accordancewith the guidelines of the Canadian Council on Animal Care for the useof experimental animals (http://www.ccac.ca/). Surgeries are performedin adult male Brown Norway rats, retired breeders, between 10-12 monthsof age (300-400 g), under general anesthesia by intraperitonealinjection of 1 ml/kg standard rat cocktail consisting of ketamine (100mg/ml), xylazine (20 mg/ml) and acepromazine (10 mg/ml).

Retrograde labeling of RGCs: For neuronal survival experiments, RGCs areretrogradely labeled with 3% DiI(1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate;Molecular Probes, Junction City, Oreg.), a fluorescent carbocyaninemarker that persists for several months without fading or leakage anddoes not interfere with the function of labeled cells. For retrogradelabeling, both superior colliculi, the main targets of RGCs in thebrain, are exposed and a small piece of gelfoam (Pharmacia and UpjohnInc., Mississauga, ON) soaked in DiI will be applied to their surface.Seven days after DiI application, the time required for labeling theentire RGC population, animals are subjected to ocular hypertensionsurgery as described below.

Ocular hypertension surgery: Unilateral and chronic elevation of IOP areinduced using a method that involves injection of a hypertonic salinesolution into an episcleral vein. All the animals involved in this studyreceive only a single saline vein injection. The eye selected for theprocedure is adapted with a plastic ring applied to the ocular equatorto confine the saline injection to the limbal plexus. A microneedle(30-50 pm in diameter) is be used to inject 50 μl of sterile 1.85 M NaClsolution through one episcleral vein. The plastic ring temporarilyblocks off other episcleral veins forcing the saline solution into theSchlemm's canal to create isolated scarring. Animals are kept in a roomwith constant low fluorescent light (40-100 lux) to stabilize circadianIOP variation.

Measurement of intraocular pressure (IOP): IOP from glaucomatous andnormal (contralateral) eyes is measured in awake animals using acalibrated tonometer (TonoPen XL, Medtronic Solan, Jacksonville, Fla.).IOP is measured every other day for the first two weeks after ocularhypertension surgery; and at least once between week 2 and week 3 afterocular hypertension surgery. Eyes become fragile after intraocularinjections in conditions of high IOP (see below), so it is advisable tolimit the number of IOP measurements as this involves putting additionalpressure on the cornea to get a reliable reading. The mean IOP (mmHg±S.E.M.) per eye is considered as the average of all IOP readingssince the onset of pressure elevation. The maximum IOP measured in eachindividual eye, glaucomatous or normal contralateral eye is defined asthe peak IOP and this value is used to estimate the mean peak IOP foreach group. The positive integral IOP is calculated as the area underthe IOP curve in the glaucomatous eye minus that of the fellow normaleye from ocular hypertension surgery to euthanasia. Integral IOPrepresents the total, cumulative IOP exposure throughout the entireexperiment.

Intraocular injection of siRNA molecules: Intraocular injections areperformed by infusing each siRNA compound at a concentration of 20 μginto the vitreous chamber of the left eye (total injection volume: 5μl). The right eye serves as contralateral control. In addition,non-operated eyes from naïve rats are used as intact controls.Intravitreal injections are performed using a 10-μl Hamilton syringeadapted with a ˜32-gauge glass needle. The needle tip is inserted intothe superior hemisphere of the eye, at the level of the pars plana, at a45° angle through the sclera into the vitreous body. This route ofadministration avoids retinal detachment or injury to eye structures,including the lens and the iris, which can release factors that induceRGC regeneration and survival. The injection is performed over a periodof 2 min and the needle is kept in place for an additional 2 min, afterwhich it is gently removed. Surgical glue (Indermill, Tyco Health Care,Mansfield, Mass., USA) will be used to seal the site of injection.

Topical Eye Drop instillation of siRNA molecules:

Daily during week 3 after IOP induction a 3 μl sample volume of 100 μgof siRNA are applied to the corneal surface bilaterally to theanesthetized animals, by a blunt pipette tip (filter tips 10 μl sterile(short)). The animals are placed in a warm environment to preventanesthesia-induced hypothermia

Quantification of surviving RGC soma: Quantification of RGC bodies isperformed in duplicate and in a masked fashion. For RGC density counts,rats are deeply anesthetized and then perfused transcardially with 4%paraformaldehyde (PFA) in 0.1 M phosphate buffer and both eyes areimmediately enucleated. Retinas are dissected and flat-mounted on aglass slide with the ganglion cell layer side up. Under fluorescencemicroscopy, DiI-labeled neurons is counted in 12 standard retinal areasas described.

IOP (intra ocular pressure) is induced and followed for 2 weeks. Then,CASP2_(—)4 or control siRNA are administered by either Eye Drop (siRNAin a 2% methyl cellulose formulation) or IVT (siRNA in PBS) as detailedin the study design in Table C27.

TABLE C27 Study Design Intravitreal injection Eye drops (5 uL) (3uL/eye) Group Treatment Dose Frequency Dose Frequency Termination 1CASP2_4 20 ug Once a week NA* NA 5 weeks after starting at 2 IOPinduction weeks after IOP induction (total 3 times during weeks 3, 4, 5)2 SiGFP 20 ug Once a week NA* NA 5 weeks after starting at 2 IOPinduction weeks after IOP induction (total 3 times during weeks 3, 4, 5)3 CASP2_4 20 ug Once after 2 100 ug Daily during 5 weeks after weeks ofIOP weeks 4 and IOP induction induction at the 5 after IOP beginning ofinduction week 3 4 siGFP 20 ug Once after 2 100 ug Daily during 5 weeksafter weeks of IOP weeks 4 and IOP induction induction 5 after IOP atthe beginning induction of week 3 5 CASP2_4 100 ug Daily during 3 weeksafter week 3 after IOP induction IOP induction 6 siGFP 100 ug Dailyduring 3 weeks after week 3 after IOP induction IOP induction*NA—non-applicable, Group size: n = 5

The results show that non-invasive delivery of siRNA formulated in a 2%methyl cellulose formulation provides neuroprotection and increasesneuronal survival.

Example 14 Rat Optic Nerve Crush (ONC) Model: Comparison of IntravitrealsiRNA Delivery to Topical Eye Drop Delivery

For optic nerve transsection the orbital optic nerve (ON) ofanesthetized rats is exposed through a supraorbital approach, themeninges severed and all axons in the ON transected by crushing withforceps for 10 seconds, 2 mm from the lamina cribrosa.

The siRNA compounds are delivered alone or in combination in 5 uL volume(20 ug/uL) as eye drops. Immediately after optic nerve crush (ONC), 20ug/10 ul test siRNA or 10 ul PBS is administered by WT to one or botheyes of adult Wistar rats. After that, siRNA is applied to the eye by EDevery other day and the levels of siRNA taken up into the dissected andsnap frozen whole retinas at 5 hours and 1 day, and later at 2 days, 4days, 7 days, 14 days and 21 days post injection is determined. Similarexperiments are performed in order to test activity and efficacy ofsiRNA administered via eye drops.

Table C28 below shows an experimental procedure to determine efficacy oftest siRNAs (siTEST1; siTEST2) alone or in combination, in the ONCmodel. Additional dosing parameters, concentrations, terminationschedules, formulations and the like are contemplated.

TABLE C28 Study Design Group (n = 8) Right eye (n = 8) Left eye (n = 8)termination 1 and 2 20 μg siTEST1 on days 0 and 7 20 μg siGFP on days 0and Day 21 (intravitreal) 7 (intravitreal) 3 and 4 20 μg siTEST1 on days10 and 20 20 μg siGFP on days 10 Day 30 (intravitreal) and 20(intravitreal) 5 and 6 PBS (eye drops) every other day PBS on days 0 and7 Day 21 starting from day 0, 3 (intravitreal) times/week) 7 and 8 50 μgsiTEST1 (eye drops) every 50 μg siGFP (eye drops) Day 10 other daystarting from day 0, 3 every other day starting times/week) from day 0,3 times/week) 9 and 10 50 μg siTEST1 (eye drops) every 50 μg siGFP (eyedrops) Day 21 other day starting from day 0, 3 every other day startingtimes/week) from day 0, 3 times/week) 11 and 12 50 μg siTEST2 (eyedrops) every 50 μg siGFP (eye drops) Day 21 other day starting from day0, 3 every other day starting times/week) from day 0, 3 times/week) 13and 14 20 μg siTEST1 + 40 μg siGFP on days 0, 7 Day 21 20 μg siTEST2 and20 (eye drops) every (eye drops) every other day other day starting fromday starting from day 0, 3 0, 3 times/week) times/week) 15 and 16 20 μgsiTEST + 40 μg siGFP on days 0 and Day 21 20 μg siTEST2 on days 0, and10 10 (intravitreal) n = 4 (intravitreal) n = 4

Results: According to the results that are obtained in this studynon-invasive delivery of siRNA compound designed for down regulation ofa target gene provides neuroprotection and increases neuronal survivalin the retina.

Tables B1-B26 disclose oligonucleotide pairs of sense and antisensenucleic acids useful in synthesizing unmodified or chemically modifiedsiRNA compounds. The tables disclose the position of the sense strandalong the mRNA for at least one variant.

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LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150050328A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1.-73. (canceled)
 74. A method of treating a patient suffering, or atrisk of suffering, from an ocular disease, an ocular disorder or anocular injury, comprising administering to the patient a therapeuticallyeffective amount of a double-stranded oligonucleotide having thestructure: (sense strand; SEQ ID NO: 9015) 5′ iB-GCCAGAAUGUGGAACUCCU 3′(antisense strand; SEQ ID NO: 9516) 3′ CGGUCUUACACCUUGAGGA 5′

wherein each of A, C, U and G is a nucleotide and each consecutivenucleotide is joined to the next nucleotide by a phosphodiester bond;wherein the sense strand comprises, counting from the 5′ terminus, anunmodified ribonucleotide at each of positions 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 and 19, a L-deoxycytidine at position18, and an inverted deoxyabasic moiety (iB) covalently attached at the5′ terminus; and wherein the antisense strand comprises, counting fromthe 5′ terminus, a 2′-O-Methyl sugar-modified ribonucleotide at each ofpositions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and an unmodifiedribonucleotide at positions 1, 3, 5, 7, 9, 10, 12, 14, 16 and 18; or apharmaceutically acceptable salt of such double-strandedoligonucleotide; and a pharmaceutically acceptable carrier.
 75. Themethod of claim 74, wherein the ocular disease, ocular disorder, orocular injury is associated with pathological abnormalities/changes inthe tissues of the visual system.
 76. The method of claim 75, whereinthe pathological abnormalities/changes comprise loss of retinal ganglioncells or retinal ganglion cell damage.
 77. The method of claim 76,wherein the loss of retinal ganglion cells or the retinal ganglion celldamage is mediated by elevated intraocular pressure (TOP).
 78. Themethod of claim 74, wherein the ocular disease, ocular disorder, orocular injury is selected from the group consisting of elevatedintraocular pressure (TOP), glaucoma, diabetic retinopathy (DR),diabetic macular edema (DME), age related macular degeneration (AMD),optic neuritis, central retinal vein occlusion, brunch retinal veinocclusion, optic nerve injury, retinopathy of prematurity (ROP),retinitis pigmentosa (RP), retinal ganglion degeneration, maculardegeneration and an optic neuropathy.
 79. The method of claim 78,wherein the ocular disease, ocular disorder, or ocular injury comprisesan optic neuropathy.
 80. The method of claim 79, wherein the opticneuropathy is selected from the group consisting of ischemic opticneuropathy (ION), hereditary optic neuropathy, Leber's hereditary opticneuropathy, metabolic optic neuropathy, neuropathy due to a toxic agent,neuropathy caused by an adverse drug reaction and neuropathy caused by avitamin deficiency.
 81. The method of claim 80, wherein the opticneuropathy comprises ischemic optic neuropathy.
 82. The method of claim81, wherein the ischemic optic neuropathy comprises anterior ischemicoptic neuropathy and/or posterior ischemic optic neuropathy.
 83. Themethod of claim 74, wherein the oligonucleotide is administered in anamount effective to down-regulate the expression of a CASP2 gene in anocular cell.
 84. The method of claim 74, wherein the oligonucleotide isadministered as a cream, a foam, a paste, an ointment, an emulsion, aliquid solution, an eye drop, a gel, spray, a suspension, amicroemulsion, microspheres, microcapsules, nanospheres, nanoparticles,lipid vesicles, liposomes, polymeric vesicles, a patch, or a contactlens.
 85. The method of claim 84, wherein the oligonucleotide isadministered as a liquid solution.
 86. The method of claim 85, whereinthe liquid solution is administered by intravitreal injection.
 87. Amethod of providing neuroprotection in a retinal cell in a patientsuffering, or at risk of suffering, from an ocular disease, an oculardisorder or an ocular injury, comprising administering to the patient atherapeutically effective amount of a double-stranded oligonucleotidehaving the structure: (sense strand; SEQ ID NO: 9015) 5′iB-GCCAGAAUGUGGAACUCCU 3′ (antisense strand; SEQ ID NO: 9516) 3′CGGUCUUACACCUUGAGGA 5′

wherein each of A, C, U and G is a nucleotide and each consecutivenucleotide is joined to the next nucleotide by a phosphodiester bond;wherein the sense strand comprises, counting from the 5′ terminus, anunmodified ribonucleotide at each of positions 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 and 19, a L-deoxycytidine at position18, and an inverted deoxyabasic moiety (iB) covalently attached at the5′ terminus; and wherein the antisense strand comprises, counting fromthe 5′ terminus, a 2′-O-Methyl sugar-modified ribonucleotide at each ofpositions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and an unmodifiedribonucleotide at positions 1, 3, 5, 7, 9, 10, 12, 14, 16 and 18; or apharmaceutically acceptable salt of such double-strandedoligonucleotide; and a pharmaceutically acceptable carrier.
 88. Themethod of claim 87, wherein the patient is suffering from an oculardisease, ocular disorder, or ocular injury selected from the groupconsisting of elevated intraocular pressure (TOP), glaucoma, diabeticretinopathy (DR), diabetic macular edema (DME), age related maculardegeneration (AMD), optic neuritis, central retinal vein occlusion,brunch retinal vein occlusion, optic nerve injury, retinopathy ofprematurity (ROP), retinitis pigmentosa (RP), retinal gangliondegeneration, macular degeneration and an optic neuropathy.
 89. Themethod of claim 88, wherein the ocular disease, ocular disorder, orocular injury comprises an optic neuropathy.
 90. The method of claim 89,wherein the optic neuropathy is selected from the group consisting ofischemic optic neuropathy (ION), hereditary optic neuropathy, Leber'shereditary optic neuropathy, metabolic optic neuropathy, neuropathy dueto a toxic agent, neuropathy caused by an adverse drug reaction andneuropathy caused by a vitamin deficiency.
 91. The method of claim 90,wherein the neuropathy comprises ischemic optic neuropathy.
 92. Themethod of claim 91, wherein the ischemic optic neuropathy comprisesanterior ischemic optic neuropathy and/or posterior ischemic opticneuropathy.
 93. The method of claim 87, wherein the oligonucleotide isadministered in an amount effective to down-regulate the expression of aCASP2 gene in an ocular cell.
 94. The method of claim 87, wherein theoligonucleotide is administered as a cream, a foam, a paste, anointment, an emulsion, a liquid solution, an eye drop, a gel, spray, asuspension, a microemulsion, microspheres, microcapsules, nanospheres,nanoparticles, lipid vesicles, liposomes, polymeric vesicles, a patch,or a contact lens.
 95. The method of claim 94, wherein theoligonucleotide is administered as a liquid solution.
 96. The method ofclaim 95, wherein the liquid solution is administered by intravitrealinjection.